CN103145868A - Derivate of low molecular weight fucosylated glycosaminoglycan and medical composition and preparation method and application thereof - Google Patents

Abstract

The invention provides a derivate of low molecular weight fucosylated glycosaminoglycan (dLFG) and a medical composition containing the dLFG or pharmaceutically acceptable salt and application of the dLFG and the medical composition for preparing medicines for treating thrombotic diseases.

Description

A low-molecular-weight glycosaminoglycan derivative, its pharmaceutical composition, its preparation method and application

technical field

The present invention belongs to the technical field of medicine, in particular to a low molecular weight fucosylated glycosaminoglycan derivative (derivate of Low molecular weight Fucosylated Glycosaminoglycan, dLFG) with anticoagulant activity, its preparation method, containing the dLFG or its pharmaceutical The pharmaceutical composition of the above acceptable salt, and the application of dLFG and its pharmaceutical composition in the preparation of drugs for the treatment of thrombotic diseases.

Background technique

Thromboembolic diseases, including ischemic stroke, coronary heart disease, and venous thromboembolism, are the main cause of death in humans. Antithrombotic drug therapy is the basic method for clinical prevention and treatment of thrombotic diseases, but antithrombotic drugs including fibrinolytic, anticoagulant, and antiplatelet drugs all have major and common defects: bleeding tendency and serious bleeding risk.

The classic anticoagulant drugs heparin (f.IIa/Xa inhibitor) and coumarin anticoagulant (VitK antagonist) were used clinically in the 1930s and 1940s, and have been the main cause of deep vein thrombosis and cardiogenic disease for more than 70 years. The cornerstone of medical therapy for stroke and postoperative anticoagulation, but the risk of bleeding and associated pharmacodynamic/pharmacokinetic deficits severely limit their clinical use. Both heparin and coumarin drugs widely inhibit serine proteases (coagulation factors) in the blood coagulation cascade, and the individual differences in drug efficacy are large and the factors affecting drug efficacy are complex. Clinical drug use requires continuous monitoring. For decades, one of the main approaches and goals of anticoagulant drug research and development is to improve the selectivity of pharmacological targets of new drugs, improve drug efficacy, and pharmacokinetic characteristics. These studies have made positive progress: among them, low molecular weight heparin (LMWH) such as enoxaparin and oral anticoagulants rivaroxaban and dabigatran are the most representative clinical applications. However, there is still a huge demand for new anticoagulants with low bleeding tendency in clinical practice.

et.al,J Biol Chem,1996,271:23973-23984)。Fucosylated glycosaminoglycan (FGAG) is a glycosaminoglycan analogue derived from the body wall or viscera of echinoderm sea cucumbers: FGAG has a main chain similar to chondroitin sulfate, and its main chain is composed of hexuronic acid aldehyde The disaccharide structural unit [→4)D-GlcUA(β1→3)D-GalNAc(β1→] composed of acid and hexosaminose is sequentially connected; there is fucosyl side chain substitution on the main chain of FGAG, generally It is believed that the side-chain sulfated fucose is linked to D-GlcUA by (α1→3) glycosidic bonds; FGAG main chain and side-chain sugar hydroxyl groups can be sulfated (Yoshida et.al, Tetrahedron Lett, 1992, 33: 4959-4962;

et.al, J Biol Chem, 1996, 271:23973-23984).

et al.,Thromb Res,2001,102:167-176;

 et.al,J Biol Chem,1996,271:23973-23984)，其抗凝活性涉及多个凝血因子靶点，其中以抑制内源性因子X酶(f.Xase,Tenase)活性最强(Sheehan&Walke,Blood,2006,107:3876-3882；Buyue&Sheehan,Blood,2009,114:3092-3100)。除抗凝活性外，FGAG还存在较广泛的药理活性，如抗炎、抗肿瘤、纤溶、调血脂活性等(Tovar et al.,Atherosclerosis,1996,126:185-195;Kariya et al.,J Biochem,2002,132(2):335-343;Borsig et al.,J Biol Chem,2007,282(20):14984-14991)，并且具有与抗凝活性矛盾的血小板活化及f.XII激活活性(Li etal.,Thromb Haemost,1988,59(3):435-439;Fonseca et al.,Thromb Haemost,2010,103(5):994-1004)，广泛的药理学作用限制了FGAG的实际应用。Natural sources of FGAG have significant anticoagulant activity (

et al., Thromb Res, 2001, 102:167-176;

et.al, J Biol Chem, 1996,271:23973-23984), its anticoagulant activity involves multiple blood coagulation factor targets, among them the activity of inhibiting endogenous factor X enzyme (f.Xase, Tenase) is strongest (Sheehan&Walke , Blood, 2006, 107:3876-3882; Buyue & Sheehan, Blood, 2009, 114:3092-3100). In addition to anticoagulant activity, FGAG also has a wide range of pharmacological activities, such as anti-inflammatory, anti-tumor, fibrinolytic, blood lipid-regulating activities, etc. (Tovar et al., Atherosclerosis, 1996, 126:185-195; Kariya et al., J Biochem,2002,132(2):335-343; Borsig et al., J Biol Chem,2007,282(20):14984-14991), and has platelet activation and f.XII activation contradictory to anticoagulant activity Activity (Li et al.,Thromb Haemost,1988,59(3):435-439; Fonseca et al.,Thromb Haemost,2010,103(5):994-1004), extensive pharmacological effects limit the actual application.

The structural modification of FGAG is one of the ways to improve its potential application value, such as the preparation of its oligomerization products, the purpose of which is to reduce its platelet and surface activation activities on the basis of retaining anticoagulant activity. Chinese Patent Publication Nos. CN101735336A and CN101724086A disclose a method for preparing oligomeric fucosylated glycosaminoglycans, by depolymerizing fucosylated glycosaminoglycans in an aqueous medium using a fourth-period transition metal ion-catalyzed peroxide depolymerization method. The oligomeric product prepared from sugar is a powerful inhibitor of endogenous factor X enzyme, has good anticoagulant and antithrombotic activity, and significantly reduces bleeding tendency, and can be used for preventing and/or treating thrombotic diseases. European patents EP 0811635A1 and EP 0408770A1 depolymerized the prototype FGAG with hydrogen peroxide to obtain depolymerization products with molecular weights ranging from 3,000 to 80,000 and 3,000 to 42,000, respectively. These products can be used for the prevention and treatment of vascular intimal hyperplasia and thrombotic diseases . These patent applications all adopt the peroxide depolymerization method to obtain the oligomerization product of FGAG. The advantage of the peroxidative depolymerization method is that it can better maintain the characteristic chemical structure and anticoagulant activity of FGAG while reducing the molecular weight of the polysaccharide. The time point at which the reaction was terminated. Fucosylated glycosaminoglycan derivatives with Δ ^4,5 unsaturated bonds at non-reducing ends have not been reported so far.

Contents of the invention

The object of the present invention is to provide a low molecular weight fucosylated glycosaminoglycan derivative (derivateof Low molecular weight Fucosylated Glycosaminoglycan, dLFG) with anticoagulant activity, its preparation method, containing the dLFG or its pharmaceutically acceptable The pharmaceutical composition of the salt, and the application of dLFG and the pharmaceutical composition thereof in the preparation of drugs for the treatment of thrombotic diseases.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

The present invention first provides a low molecular weight fucosylated glycosaminoglycan derivative (dLFG) and a pharmaceutically acceptable salt thereof. The constituent monosaccharides of the dLFG include hexuronic acid, hexosamine, deoxyhexose and sulfate esters of these monosaccharides. Wherein, the hexuronic acid is D-glucuronic acid (D-GlcUA) and Δ ^4,5 -hexuronic acid (4-deoxy-threo-hex-4-enpyranuronic acid, ΔUA), The hexosamine is D-acetylgalactosamine (2-N-acetylamino-2-deoxy-D-galactose, D-GalNAc) or its terminal reduction product, and the deoxyhexose is L-rock Alcose (L-Fuc).

In terms of molar ratio, the composition ratio range of the dLFG monosaccharide and -OSO ₃ ^- is: hexuronic acid: hexosamine: deoxyhexose: sulfate group = 1: (1 ± 0.3): (1 ± 0.3): (3.0±1.0). Generally speaking, in the hexuronic acid contained in the dLFG of the present invention, the percentage of ΔUA is not less than 2.5% in terms of molar ratio.

The molecular weight of the dLFG of the present invention can be detected and calibrated by using a high-efficiency gel permeation chromatograph and a laser small-angle light scattering instrument (HPGPC-LALLS); the series dLFG with a calibrated molecular weight is used as a standard product to make a standard curve, and a UV detector is used in conjunction High performance gel chromatography (HPGPC) with (UVD) and/or differential detector (RID) routinely detects the molecular weight of the dLFG.

In terms of weight average molecular weight (Mw), the molecular weight of the dLFG in the present invention ranges from 3kD to 20kD; in a preferred embodiment of the present invention, the Mw of the dLFG ranges from about 5kD to 12kD.

The polydispersity index (polydispersity index, PDI) of the dLFG in the present invention is generally between 1.0 and 1.8, and the so-called PDI refers to the ratio of the weight average molecular weight Mw to the number average molecular weight Mn of the dLFG. In a preferred embodiment of the present invention, the PDI value of the dLFG is between 1.1 and 1.5.

Further, the low-molecular-weight glycosaminoglycan derivatives of the present invention are a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),

In formula (I):

n is an integer with an average value of about 2-20; in preferred compounds of the invention, n is an integer with an average value of about 4-12.

-D-GlcUA-β1-, is -D-glucuronic acid-β1-yl-;

-D-GalNAc-β1-, is -2-deoxy-2-N-acetylamino-D-galactosyl-β1-yl-;

L-Fuc-α1-, is -L-fucose-α1-yl-;

R are independently -H or -SO ₃ ^- ;

R' is -OH, C1-C6 alkoxy, C7-C12 aryloxy;

_R is the non-reducing end of the homologous glycosaminoglycan derivative of the present invention, and its structure is a group shown in formula (II) or (III),

In formula (II), (III),

ΔUA-1-, is Δ ^4,5 -hexuronic acid-1-yl (4-deoxy-threo-hex-4-enpyranuronic acid-1-yl);

R, R' are as defined above.

In the mixture of homologous glycosaminoglycan derivatives described in the present invention, in terms of molar ratio, the ratio of R ₁ to the compound of formula (II) and R ₁ to formula (III) is not less than 2:1. In a preferred embodiment of the present invention, in the mixture of homologous glycosaminoglycan derivatives, the molar ratio of non-reducing terminal R ₁ to the structures of formula (II) and formula (III) is not lower than about 4:1.

R ₂ is a group shown in formula (IV) or (V),

In the formula, R and R' are as defined above;

R ₃ is carbonyl, hydroxyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, C6-C12 aryl, substituted or unsubstituted five-membered or six-membered nitrogen-containing heterocyclic group, or -NHR ₄ ; where ,

R ₄ is a substituted or unsubstituted linear or branched C1-C6 hydrocarbon group, a substituted or unsubstituted C7-C12 aryl group, a substituted or unsubstituted heteroatom-containing heterocyclic aryl group.

In a preferred embodiment of the present invention, said R ₃ is -CHO or -CH ₂ OH.

The dLFG described in the present invention is the depolymerization product obtained by β-elimination (β-elimination) of fucosylated glycosaminoglycan (FGAG) extracted from the body wall and/or viscera of Echinodermata and Holothurian, and the resulting The reducing end of the FGAGs depolymerization product is reduced or reduced aminated product. The FGAG derived from the echinoderm generally has the following characteristics:

et.al.,J Biol Chem,1996,271:23973-23984;

et al.,Thromb Res,2001,102:167-176;

et.al,J Biol Chem,1996,271:23973-23984;Tovaret al.,Atherosclerosis,1996,126:185-195;Kariya et al.,J Biochem,2002,132(2):335-343;Borsig et al.,J Biol Chem,2007,282(20):14984-14991。(1) The FGAG is extracted from the body wall or viscera of Echinodermata and sea cucumbers, and its extraction and preparation methods are well known to researchers in the field, and can be found in literature, such as Yoshida et.al., Tetrahedron Lett ,1992,33:4959-4962;

et.al., J Biol Chem, 1996, 271:23973-23984;

et al., Thromb Res, 2001, 102:167-176;

et.al, J Biol Chem, 1996,271:23973-23984;Tovar et al.,Atherosclerosis,1996,126:185-195;Kariya et al.,J Biochem,2002,132(2):335-343;Borsig et al., J Biol Chem, 2007, 282(20):14984-14991.

(2) The constituent monosaccharides of the FGAG include D-glucuronic acid (GlcUA), D-N-acetyl-2-amino-2-deoxygalactose (GalNAc) and L-fucose (Fuc). Sulfate substituents may be present on sugar groups;

(3) The main chain of the FGAG contains [-4-D-GlcUA-β1-3-D-GalNAc-β1-] repeating structural unit, and the Fuc sugar group is connected to the main chain GlcUA sugar group in the form of a side chain.

Generally speaking, described FGAG has the structure shown in formula (VI):

In formula VI,

n is an integer with an average of about 40 to 90;

R are independently -OH or -OSO3-;

R ₅ is -H or D-GlcUA-β1-;

R ₆ is -OH, -4-D-GalNAc or a sulfate ester thereof.

The Echinodermata sea cucumber class animal described in the present invention may include but not limited to:

Apostichopus japonicus;

Actinopyga mauritiana;

Actinopyga miliaris;

Haitian melon Acaudina molpadioides;

Bohadschia argus;

Red-bellied sea cucumber Holothuria edulis;

Black milk sea cucumber Holothuria nobilis;

Yuzu sea cucumber Holothuria leucospilota;

Chinese sea cucumber Holothuria sinica;

Dangpi sea cucumber Holothuria vagabunda;

American ginseng Isostichopus badionotus;

Brazilian ginseng Ludwigothurea grisea;

Green sea cucumber Stichopus chloronotus;

Thelenota ananas;

Thelenota anax;

In a preferred embodiment of the present invention, the Echinodermata sea cucumber class animals may include sea cucumbers, sea cucumbers and plum blossoms.

Another object of the present invention is to provide a method for preparing dLFG of the present invention and a pharmaceutically acceptable salt thereof, the preparation method generally comprising the following steps:

(1) Using fucosylated glycosaminoglycans (FGAG) derived from echinoderms as raw materials, all or part of the free carboxyl groups on the GlcUA contained in the FGAG are converted into carboxylate groups through an esterification reaction, thereby obtaining FGAG carboxylate derivatives;

(2) The FGAG carboxylate derivative obtained in step (1) is in a non-aqueous solvent, in the presence of an alkaline reagent, through the β-elimination reaction of the carboxylate group to obtain a non-reducing terminal Δ ^4,5 -hexose Low molecular weight glycosaminoglycan derivatives (dLFG) of uronic acid (ΔUA);

(3) The dLFG obtained in step (2) may optionally be subjected to reduction treatment at the reducing end, thereby obtaining dLFG with the reduced end.

(4) The dLFG obtained in step (2) and step (3) can optionally convert carboxylate and/or amide groups into free carboxyl groups by alkaline hydrolysis.

The present invention found that the fucosylated glycosaminoglycan FGAG of natural origin is relatively stable in alkaline aqueous solution. Under severe reaction conditions such as elevated temperature and enhanced alkalinity, FGAG may be accompanied by β-elimination reaction. The destruction of the characteristic structure of glycosylated glycosaminoglycans, including fucose side chains, sugar ring structures of the main chain, and cleavage of sulfate groups, etc., so the reaction products are difficult to predict, and the chemical structures of the reaction products are complex and difficult to confirm. Obviously, , it is difficult to obtain FGAG depolymerization products with uniform structure by direct alkali treatment.

In order to achieve the purpose of depolymerizing FGAG by the β-elimination method, the present invention establishes a β-elimination method that selectively esterifies the free carboxyl groups of hexuronic acid, and then realizes mild conditions and maintains the integrity of the characteristic structure of glycosylated glycosaminoglycans Depolymerization. In the present invention, the "integrity of characteristic structure" means: except that the molecular weight is reduced and the chemical structure modification of terminal sugar groups (including reducing terminal and non-reducing terminal sugar groups) may exist, the basic chemical structural characteristics of the depolymerization product are equivalent to Undepolymerized polysaccharides, including the composition and ratio of monosaccharides, the connection mode of repeating structural units, the number and type of sulfate groups, etc.

Among the present invention, the preparation method of FGAG carboxyl esterification product is:

(1) FGAG is converted into a quaternary ammonium salt by an ion exchange method, for example, the FGAG aqueous solution is passed through a H ⁺ type cation exchange resin to convert the neutral FGAG salt into a H ⁺ type FGAG;

(2) Titrate/neutralize the obtained H ⁺ type FGAG solution with a quaternary ammonium alkali solution to obtain a FGAG quaternary ammonium salt solution, and freeze-dry the resulting solution to obtain the FGAG quaternary ammonium salt. The quaternary ammonium base may include but not limited to tetrabutylammonium hydroxide, dodecyltrimethylammonium hydroxide, tetramethylammonium hydroxide, tetrapropylammonium hydroxide, tetraethylammonium hydroxide, benzyl trimethylammonium hydroxide Methylammonium hydroxide, benzyltriethylammonium hydroxide, etc.

(3) FGAG quaternary ammonium salt reacts with stoichiometric halogenated hydrocarbons in an aprotic solvent such as dimethylformamide (N,N-Dimethylform-amide, DMF), and the reaction product is separated and purified to obtain FGAG carboxyl ester derivatives (quaternary ammonium salt, which can be used directly for further β-elimination reactions). The hydrocarbon group in the halogenated hydrocarbon includes but not limited to C1-C6 linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic hydrocarbon group; substituted or unsubstituted C7-C12 aromatic hydrocarbon group and the like.

Another invention patent application of the applicant, the application number is 201110318704.X (publication number is CN102329397A) introduces the preparation method of FGAG carboxyl ester derivatives.

During the preparation of low-molecular-weight heparin, carboxylate derivatives of unfractionated heparin can undergo β-elimination reaction in alkaline aqueous solution, thereby obtaining low-molecular-weight heparin (LMWH) with ΔUA at the end. The experimental research of the present invention surprisingly found that under similar conditions such as NaOH aqueous solution, the FGAG carboxyl esterification product can be completely hydrolyzed into the prototype FGAG, with little or almost no β-elimination reaction. Therefore, the present invention focuses on the non-aqueous solvent system that can carry out the β-elimination reaction of FGAG carboxyl ester derivatives.

In the preparation method of dLFG and its pharmaceutically acceptable salts described above in this specification, the non-aqueous solvent described in step (2) can be ethanol, methanol, dimethylformamide, dimethyl sulfoxide, _CH2 Cl ₂ , CHCl ₃ or their mixed solvents. The alkaline reagent is optionally NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo or mixtures thereof.

One of the key technical difficulties to be solved in the β-elimination reaction of FGAG carboxyl ester derivatives using non-aqueous solvents is that the solubility of FGAG carboxyl ester derivatives in non-aqueous solvents is extremely poor, thus seriously affecting the desired β-elimination reaction carried out. The scheme that the present invention improves the solubility of FGAG carboxyl ester derivatives in non-aqueous solvents is to further convert it into quaternary ammonium salts, thus realizing the FGAG carboxyl ester derivatives that can meet the needs of β-elimination reactions in non-aqueous solvents Solubility.

When using a single lower alcohol/ketone such as ethanol as a solvent, its solubility to the FGAG carboxyl esterification product quaternary ammonium salt is relatively low, and other non-aqueous solvents such as dimethylformamide (N,N-Dimethyl-formamide ,DMF), because the solubility of NaOH as a catalyst is very low, the reaction efficiency can be greatly limited. For this reason, in the design of the present invention, on the one hand, adopt C1-C4 sodium alkoxide as alkaline reagent in order to improve the concentration of alkaline reagent in non-aqueous solvent, be to select to adopt mixed solvent system as the solvent of β-elimination reaction on the other hand system, the reaction system is carried out through the following steps: dissolving the quaternary ammonium salt of the FGAG carboxyl ester derivative in a suitable non-aqueous solvent such as DMF; dissolving NaOH or other suitable base catalysts in anhydrous lower alcohol; dissolving the FGAG The quaternary ammonium salt solution of the carboxyl esterification product is mixed with the alkali catalyst solution, thereby obtaining a clear β-elimination reaction system.

The alkaline reagent includes but not limited to NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo or mixtures thereof. The preferred alkaline reagent of the present invention is sodium ethylate, and the preferred technical method is to add stoichiometric sodium metal in dry anhydrous ethanol solution, thereby preparing sodium ethylate-ethanol solution.

In the β-elimination reaction solution, the concentration of the quaternary ammonium salt of the FGAG carboxyl ester derivative is generally 1-150 mg/ml, and the concentration of the alkali catalyst is generally 0.1-100 mmol/L. In the β-elimination reaction solution, the β-elimination reaction of the carboxyl esterification product of FGAG can proceed smoothly, and generally the reaction is complete at room temperature for 0.1-8 hours. After the β-elimination reaction is completed, neutralize the reaction solution with an acid (such as hydrochloric acid) to obtain the dLFG product of carboxyl esterification; or, without changing the alkaline condition of the reaction solution, add an appropriate amount of water to the reaction solution, room temperature The carboxylate groups contained in the depolymerized dLFG derivatives can be completely hydrolyzed by keeping the reaction solution for about 0.5-1 hr. At this time, the dLFG products containing free carboxyl groups can be obtained by neutralizing the reaction solution with acid.

In general, FGAG carboxyl ester preparation and its β-elimination depolymerization can be adopted:

FGAG quaternary ammonium salt is obtained by neutralizing FGAG neutral salt through H+ cation exchange resin column and quaternary ammonium alkali titration, and then reacted with aprotic solvent, halogenated hydrocarbon, room temperature or heating (carboxyl esterification) to obtain FGAG carboxyl esterification The product (quaternary ammonium salt) is then dissolved in a non-aqueous solvent, added with a strong base, and reacted at room temperature or under heating (β-elimination) to finally obtain dLFG (depolymerization product of FGAG’s β-elimination method).

Theoretically, when the carboxyl esterification rate on FGAG is about 5%-30%, the molecular weight of the obtained product can be between about 3kD-20kD by depolymerization according to the β-elimination method of the present invention. In the actual reaction, in view of the influence of experimental conditions on the degree of β-elimination reaction, the FGAG carboxyl esterification product with a carboxyl esterification rate of about 10% to 60% can be depolymerized by the β-elimination reaction to obtain a Mw of about 3kD~ 20 kD dLFG product.

The dLFG obtained by the β-elimination reaction of the present invention has a reducing end, and its reducing end is mainly -4-D-N-acetyl-2-amino-2-deoxygalactose (-4-D-GalNAc). In the present invention, the reducing end can optionally be terminally modified by a reduction reaction, which may include but not limited to:

(1) Reducing the reducing end to sugar alcohol in the presence of a reducing agent such as sodium borohydride. Taking -4-D-N-acetyl-2-amino-2-deoxygalactose as an example, the reaction process is:

In the formula, R is -OH or -OSO ₃ ^￣ .

(2) Reductive amination of the reducing end in the presence of an organic amine, the reaction is that the organic amine reacts with the 1-position aldehyde of the terminal sugar group to form a Schiff base, which is reduced to a secondary amine in the presence of a reducing agent. Taking the reaction of terminal-4-D-N-acetyl-2-amino-2-deoxygalactose with ammonium bicarbonate and primary amine as an example, the reaction process is as follows:

In the formula, R ₄ is H, substituted or unsubstituted straight or branched C1-C6 hydrocarbon group, substituted or unsubstituted C7-C12 aryl group, substituted or unsubstituted heteroatom-containing heterocyclic group, said hetero Atoms include but are not limited to O, N, S.

(3) In the presence of a reducing compound, the reducing end is reductively alkylated to obtain a reductively alkylated derivative. Taking the reaction of terminal-4-D-N-acetyl-2-amino-2-deoxygalactose with pyrazolone compound reducing agent as an example, the reaction process is as follows:

Reduction of the reducing end of an aldose, reductive amination and reductive alkylation are well known to those skilled in the art. In the present invention, after the reducing end of the dLFG obtained by the β-elimination method is treated by the reduction reaction, the terminal sugar groups shown in formula (IV) and formula (V) can be obtained, wherein R ₃ can be C1-C6 alkane Oxygen, C1-C6 alkoxycarbonyl, C6-C12 aryl, substituted or unsubstituted five-membered or six-membered nitrogen-containing heterocyclic group, or -NHR ₄ (R ₄ is a substituted or unsubstituted straight or branched chain C1-C6 hydrocarbon group, substituted or unsubstituted C7-C12 aryl group, substituted or unsubstituted heteroatom-containing heterocyclic aryl group).

The reaction product dLFG of the present invention can be purified by methods known in the art (Chinese patent application publication number is CN101735336A), such as removing impurities such as small molecular salts by dialysis or ultrafiltration, or by gel chromatography or DEAE ion Further purification by exchange chromatography, etc.

During the dialysis impurity removal process, a dialysis membrane or an ultrafiltration membrane package with a suitable molecular weight cut-off can be selected according to the molecular weight of the target dLFG, preferably with a molecular weight cut-off of 1000 Da. The dialysis time needs to be determined according to specific treatment conditions, usually not less than 6 hours.

The dLFG product of the present invention can also be prepared into a single salt form by cation exchange, such as alkali metal, alkaline earth metal salt and organic ammonium salt. In a preferred embodiment of the present invention, the single salt form of dLFG is sodium salt, potassium salt or calcium salt.

The salt-forming process of the dLFG product can be exchanged into the hydrogen form first, and then neutralized with the corresponding base to obtain the corresponding salt of dLFG; the dynamic ion-exchange salt-forming method can also be used to exchange the salt directly on the column, among which Strongly acidic cation exchange resin is used. Resin column pretreatment, sample loading and elution can be carried out by conventional methods.

The dLFG of the present invention is prepared from FGAG derived from echinoderm as a reaction starting material through β-elimination reaction. As mentioned above, in the present invention, the source animal of the starting material FGAG can be selected from but not limited to Apostichopus japonicus, Radix radix alba, Radix radix black wrinkle, Haitian melon, Radix chinensis, Red Belly sea cucumber, black milk sea cucumber, jade foot sea cucumber, Chinese sea cucumber, dangpi sea cucumber, American meat ginseng, Brazilian ginseng, green sea cucumber, plum ginseng, giant plum ginseng. In a preferred embodiment of the present invention, the source animals include sea cucumber, yuzu sea cucumber and plum flower ginseng.

The structural feature of the FGAG derived from Echinodermata Holothuria in the present invention is the presence of GlcUA, GalNAc and fucose or its sulfate ester in an approximately equimolar ratio (about 1:1±0.3). Different species of sea cucumbers and their tissue sources, or differences in extraction methods can lead to differences in the composition ratio of monosaccharides and the degree of sulfation of polysaccharides in FGAG, and these differences do not affect the basic structural characteristics of FGAG. Apparently, those skilled in the art can understand that fucosylated glycosaminoglycans derived from other species of sea cucumbers that conform to the basic structural characteristics of FGAG can be used to obtain the dLFG derivatives of the present invention.

The dLFG of the present invention has potent anticoagulant activity, and its drug concentration for doubling the activated partial thromboplastin time (APTT) of human quality control plasma (the drug concentration for prolonging APTT by 1 time) is not higher than 9 μg/mL. The study of the present invention confirms that the dLFG has significant activity of inhibiting endogenous factor X enzyme (f.Xase, Tenase), and has heparin cofactor II (HC-II) dependent antithrombin (f.IIa) activity . Because f.Xase is the last target of the endogenous coagulation pathway in the coagulation cascade, it is the rate-limiting step of various experimental coagulation processes (Buyue&Sheehan, Blood, 2009,114:3092-3100); it has HC-II dependent Dermatan sulfate with antithrombin activity has been clinically used in antithrombotic therapy, therefore, dLFG has the potential of clinical application in the treatment of thromboembolic diseases.

The dLFG of the present invention has no platelet activation activity; in addition, the present invention is surprised to find that the dLFG of the present invention does not have the activity of activating f.XII compared with its natural source FGAG, and does not affect the f.XII-kallikrein system , will not cause a decrease in blood pressure in experimental animals.

The non-reducing end of the dLFG of the present invention may contain ΔUA sugar groups, and the presence of unsaturated double bonds in ΔUA makes it have a maximum ultraviolet absorption (UV, λmax) at about 232-238nm. The qualitative and quantitative analysis method of the method is of great value, especially when the high performance liquid gel chromatography (HGPC) method is used to analyze the dLFG content of the sample, a UV detector with high sensitivity can be used for detection, so it is especially suitable for sample quality control, The establishment of technical methods related to dLFG content analysis such as blood drug concentration analysis.

The dLFG of the present invention has definite anticoagulant activity, and therefore has antithrombotic application value. dLFG has good water solubility, so it is easy to prepare a solution-type preparation or a freeze-dried product thereof. As a polysaccharide component, its oral bioavailability is limited, so it is preferably prepared as a parenteral dosage form, and its formulation preparation can be carried out according to well-known technical methods in the art.

Therefore, another object of the present invention is to provide a pharmaceutical composition comprising the dLFG of the present invention and pharmaceutically acceptable excipients.

The dLFG of the present invention has potent anticoagulant activity, so it can be used for the prevention and treatment of different degrees of thrombotic diseases, such as thrombotic cardiovascular disease, thrombotic cerebrovascular disease, pulmonary vein thrombosis, peripheral vein thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc. Therefore, the present invention can provide the application of the composition in the preparation of medicines for treating and preventing cardiovascular diseases.

The dLFG of the present invention has potent anticoagulant activity, so it can be used for the prevention and treatment of different degrees of thrombotic diseases, such as thrombotic cardiovascular disease, thrombotic cerebrovascular disease, pulmonary vein thrombosis, peripheral vein thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc. Therefore, the present invention can provide the application of the composition in the preparation of medicines for treating and preventing cardiovascular diseases.

The applicant is the first to study and establish the β-elimination depolymerization method of FGAG. The applicant found in the course of experimental research that the prototype FGAG is relatively stable in alkaline solution and is not prone to beta-elimination reaction. Carboxyl esterification followed by β-elimination is an optional technical method, but due to the fucosyl side chain substitution of FGAG, its polysaccharide structure is more complex. On the one hand, its complex steric hindrance can affect a variety of chemical functional groups On the other hand, a variety of chemical reaction conditions can affect the stability of side chain fucosyl glycosidic bonds. The present invention surprisingly finds that the FGAG carboxyl esterification product cannot undergo β-elimination reaction in alkaline aqueous solution like the heparin esterification product. In fact, in alkaline aqueous solution, the ester groups of FGAG carboxyl esterification products are easily hydrolyzed, and almost no β-elimination reaction occurs, which is significantly different from the properties of typical glycosaminoglycan compounds such as heparin.

The present invention successfully obtains a fucosylated glycosaminoglycan depolymerization product with a non-reducing end having a ^Δ4,5 unsaturated bond through the β-elimination reaction of the carboxyl esterification product of FGAG in a non-aqueous solvent.

By adopting the technical method of the present invention, depolymerized fucosylated glycosaminoglycan derivatives (dLFG) in the desired molecular weight range can be obtained. Physicochemical and spectroscopic chemical structure analysis showed that the basic structure remained stable except for the Δ ^4,5 unsaturated bond at the reducing end.

When adopting the method of the present invention to prepare LFG, the molecular weight of the reaction product can be effectively controlled by controlling the carboxyl esterification and/or amidation ratio; since the non-reducing end of the reaction product has a Δ ^4,5 unsaturated bond, the resulting dLFG is about 232-240nm This property can be used for quantitative detection of products, so it is beneficial to establish technical methods related to content analysis such as chemical reaction control, product quality analysis, and blood drug concentration detection.

Pharmacological experimental research results show that the dLFG prepared by the method of the present invention can have significant anticoagulant activity, which prolongs the activated partial thrombin time (APTT) of human quality control plasma, inhibits endogenous factor X enzyme (f.Xase , Tenase) and heparin cofactor II (HC-II)-dependent antithrombin (anti-f.XIIa) activity are basically consistent with those of peroxidative depolymerization products of similar molecular weight, indicating that it has good potential applications value. Compared with the peroxidative depolymerization products, the advantages of the dLFG of the present invention are as follows: firstly, it has a non-reducing end with a specific structure, and has an ultraviolet absorption of about 232-240nm at λmax, and its quality controllability is significantly improved; , the preparation process has good controllability.

Further studies of the present invention have found that the dLFG of the present invention does not have platelet activation and coagulation factor XII (f.XII) activation activity under the effective dose of anticoagulant, thus can avoid platelet activation and f.XII-kallikrein system activation A series of adverse reactions caused; Compared with heparin drugs with equivalent antithrombotic dosage, the low molecular weight FGAG of the present invention can further reduce bleeding tendency, and has the value of treating and/or preventing thrombotic diseases.

Description of drawings

Figure 1 is the HPGPC spectrum of TAG and dLFG-1A;

Figure 2(a) is the ¹ H NMR spectrum of TAG;

Figure 2(b) is the ¹ H NMR spectrum of dLFG-1A;

Figure 3 is the ¹³ C NMR spectrum of TAG and dLFG-1A;

Figure 4 is the ¹ H- ¹ H COZY NMR spectrum of TAG and dLFG-1A;

Figure 5(a) is the ¹ H- ¹ H ROESY spectrum of TAG;

Figure 5(b) is the ¹ H- ¹ H TOCSY spectrum of TAG;

Figure 5(c) is the ¹ H- ¹ H ROESY spectrum of dLFG-1A;

Figure 5(d) is the ¹ H- ¹ H TOCSY spectrum of dLFG-1A;

Figure 6 is the ¹ H- ¹³ C HSQC spectrum of dLFG-1A;

Fig. 7 is the ¹ H NMR spectrogram of AJG, LGG and HNG;

Fig. 8 is the ¹ H NMR spectrogram of HEG and dHEG;

Figure 9 is the ¹ H NMR spectrum of dLFG-2A;

Figure 10 is the dose-effect relationship of the antithrombin activity of dLFG-1E dependent on HC-II;

Figure 11 is the dose-effect relationship of dLFG-1E inhibiting f.Xase activity;

Fig. 12 is a flow chart of the basic steps of depolymerization by β-elimination method of FGAG.

Detailed ways:

The substantive content of the present invention will be described in detail below with the following examples in conjunction with the accompanying drawings, but the scope of the present invention will not be construed in any way.

【Example 1】

Preparation of low molecular weight fucosylated glycosaminoglycan derivatives (dLFG) derived from Thelenota ananas:

1.1 Materials

Prunus ginseng (Thelenota ananas Jaeger), commercially available, eviscerated and dried body wall; benzethonium chloride, benzyl chloride, tetrabutylammonium hydroxide (TBA), N,N-dimethylformamide (DMF), hydroxide Reagents such as sodium, sodium chloride and ethanol are commercially available analytical reagents.

1.2 Method

(1) Extraction and preparation of FGAG (FGAG from Thelenota ananas, TAG) from Plum ginseng: Take 300g of dried body wall of Plum ginseng, refer to the method (Kariya et al., J Biol Chem, 1990, 265(9): 5081-5085 ) to prepare TAG, the yield is about 1.5%, the purity is 98% (HPGPC, area normalization method), and the weight average molecular weight (Mw) is 65,890Da.

(2) Preparation of TAG quaternary ammonium salt: get step (1) gained TAG1.2g and place it in a beaker, add 40mL deionized water to make it dissolve; titrate with 75mg/mL benzethonium chloride solution during stirring, the solution has white color immediately Precipitation was formed, after the titration was completed, centrifuged, the precipitate was washed three times with deionized water, and finally the precipitate was vacuum-dried at room temperature for 24 hours to obtain 2.68 g of FGAG ammonium salt.

(3) Preparation of TAG benzyl esterification: Place the TAG quaternary ammonium salt obtained in step (2) into a round bottom flask, add 27 mL of DMF to dissolve, add 13.5 mL of benzyl chloride, and react with stirring at 35°C for 25 h under the protection of N ₂ . After the reaction, add 35 mL of saturated NaCl to the reaction solution, add 300 mL of absolute ethanol, centrifuge at 3500 rpm for 20 min, and remove the supernatant. The precipitate was washed three times with 200 mL of 1:9 (v/v) saturated NaCl-absolute ethanol, dissolved in 100 mL of deionized water, dialyzed with a 3500 kD dialysis bag for 24 hours, the dialysate was concentrated and freeze-dried to obtain FGAG benzyl ester, ¹ H-NMR Determine its degree of esterification as 72%.

(4) Preparation of TAG benzyl ester tetrabutylammonium salt: TAG benzyl ester obtained in the water-soluble step (3), ion exchange method (Dowex/r50w×850-100(H), 60×3cm) into hydrogen form, conductivity meter Under monitoring, titrate with 0.4M tetrabutylammonium hydroxide solution to convert all the sulfate and unesterified carboxyl groups into ammonium salts. The resulting solution was freeze-dried to obtain 1.326 g of tetrabutylammonium salt of TAG benzyl ester.

(5) Depolymerization by β-elimination method: Dissolve 800 mg of TAG benzyl ester tetrabutylammonium salt obtained in step (4) in 8.0 mL of DMF, add 0.8 mL of tri-n-butylamine, react at 60 ° C for 24 hr, and add to the solution Add 1:9 (v/v) saturated NaCl and 80 mL of absolute ethanol, and centrifuge at 3500 rpm for 15 min to obtain a precipitate. Add 8mL of 0.1M NaOH to the precipitate, react at 30°C for 40min to hydrolyze the residual carboxylate, adjust the pH to neutral with 0.1MHCl, 80mL of absolute ethanol, and centrifuge at 3500rpm for 15min to obtain the precipitate. Dissolve the resulting precipitate in 8mL H ₂ O, perhydrogen ion exchange resin column (Dowex/r50w×850-100(H), 60×3cm), adjust the pH to neutral with 0.1M NaOH, put it in a 1kD dialysis bag, and dialyze with deionized water After 24 hours, about 310 mg of the β-elimination depolymerization product dLFG-1A was obtained after freeze-drying.

(6) Physicochemical properties, monosaccharide composition and spectral detection of TAG and its β-elimination depolymerization product dLFG-1A

Molecular weight: detected by high performance gel chromatography (HPGPC). The detection conditions are Agilent technologies1200series series chromatograph, Shodex Ohpak SB-804HQ gel chromatography column, column temperature is 35°C; mobile phase is 0.1M sodium chloride, flow rate is 0.5mL/min; Agilent1100 RID and UVD combined detection. The standard curve was drawn with the series of FGAG with calibrated molecular weight, and the molecular weight and distribution were calculated by GPC.

-OSO ₃ ^- /-COO ^- molar ratio: Conductometric detection;

Optical rotation: measured according to the Chinese Pharmacopoeia (2010 edition) two appendix VI E method;

Intrinsic viscosity: according to the Chinese Pharmacopoeia (2010 edition) two appendix VI G method, measured by Ubbelohde viscometer.

Monosaccharide composition detection: Elson-Morgon method to detect acetylgalactosamine (D-GalNAc) content, carbazole method to detect glucuronic acid (D-GlcUA) content (Zhang Weijie, Glycocomplex Biochemical Research Technology (Second Edition), Zhejiang : Zhejiang University Press, 1999,19-20); the content of 4,5 unsaturated glucuronic acid residues (ΔUA) is based on the integral of H4 in ¹ H NMR compared to the integral of acetylgalactosamine (D-GalNAc) methyl Calculate; Swiss Bruker company AVANCE AV500 superconducting nuclear magnetic resonance instrument (500MHz) detects NMR spectrogram (detection condition, solvent D ₂ O, 99.9Atom%D (Norell company); Internal standard, trimethylsilyl-propionic acid (TSP-d4); temperature 300K).

Ultraviolet absorption spectrum (UV) detection: the solution obtained by 0.855mg/mL dLFG was scanned in the wavelength range of 190-400nm on Shimadzu UV-2450.

1.3 Results

The physical and chemical properties and monosaccharide composition of TAG and its depolymerization product dLFG-1A are shown in Table 1. The HPGPC chromatograms of TAG and dLFG-1A are shown in Figure 1.

The test results showed that, compared with TAG, the molecular weight and intrinsic viscosity of dLFG-1A were significantly lower.

The results of monosaccharide composition detection showed that the ratios of hexosamine, hexuronic acid (UA, the sum of GlcUA and ΔUA) and deoxyhexose (Fuc) of TAG and dLFG-1A were basically stable. It can be seen from the ¹ H NMR spectrum described later that TAG does not contain ΔUA, and the molar ratio of ΔUA contained in dLFG-1A to GalNAc contained in it is about 0.18:1 (in terms of molar ratio, ΔUA accounts for the total hexuronic acid aldehyde about 7.5% of acid).

Table 1 Detection results of physical and chemical parameters and monosaccharide composition of FGAG and dLFG-1A derived from plum blossom ginseng

The UV spectrophotometer scans in the wavelength range of 190nm to 400nm, dLFG has a maximum ultraviolet absorption λmax236nm, which is consistent with the existence of ΔUA unsaturated bonds.

Figure 2 of the present invention shows the ¹ H NMR spectrum of TAG and dLFG-1A; Figure 3 shows the ¹³ C NMR spectrum of TAG and dLFG-1A; Figure 4 shows ^{the 1} H- ¹ of TAG and dLFG-1A H COZY NMR spectrum; Figure 5(a) shows the ¹ H- ¹ H ROESY spectrum of TAG; Figure 5(b) shows ^{the 1} H- ¹ H TOCSY spectrum of TAG; Figure 5(c) shows the dLFG ¹ H- ¹ H ROESY spectrum of -1A; Figure 5(d) shows the ¹ H- ¹ H TOCSY spectrum of dLFG-1A; Figure 6 shows the ¹ H- ¹³ C HSQC spectrum of dLFG-1A.

For the signal assignment of the ¹ H NMR of TAG and its correlation spectrum, reference may be made to the Chinese Patent Publication No. CN102247401A that the applicant has applied for.

In the TAG ¹ HNMR spectrum, three sets of strong signal peaks can be seen in the range of 5.2-5.7ppm. These are the hydrogen signals of different types of sulfated α-fucose end groups, and the signal at about 5.6ppm is L-fucose The terminal hydrogen signal of sugar-2,4-disulfate group (Fuc2S4S), about 5.30-5.39ppm peaks are L-fucose-3-sulfate group (Fuc3S) and L-fucose (Fuc4S)- 4-Sulfate terminal hydrogen signal.

The main chain GlcUA and GalNAc terminal β-hydrogen signals are located at about 4.4-4.6 ppm. About 1.0-1.3ppm and 1.9-2.0ppm are respectively the methyl proton signal peaks on the Fuc methyl group and the GalNAc acetyl group. The sugar ring hydrogen at the sulfate ester substituent appears in the range of about 4.2-4.8ppm, and the signal at about 3.6-4.6ppm is the superposition of the sugar ring hydrogen at the non-sulfate substituent.

Compared with the TAG spectrum, new signal peaks appear at about 5.76 and 5.82ppm in the ¹ H NMR spectrum of dLFG-1A, and these signals can be attributed to the 4-H feature of ΔUA according to its ¹ H NMR correlation spectrum Signal.

The ¹ H- ¹ HCOSY spectrum and TOCSY spectrum of dLFG-1A clearly showed the coupling correlation among the H4, H3, H2 and H1 proton signals of ΔUA. ¹ H- ¹ H ROESY spectrum showed that Fuc was linked to GlcUA and ΔUA by α(1,3) glycosidic bonds. In addition, compared with the terminal hydrogen signal of Fuc linked to GlcUA, the terminal hydrogen signal of the same type of Fuc linked to ΔUA appears at a higher field (see Fuc2S4S terminal hydrogen in Figure 2(a), 2(b) The position of the hydrogen signal of the terminal group of Fuc2S4S connected to ΔUA).

In the ¹³ C-NMR spectrum (with TMS as the external standard), the C1 peaks of GlcUA and GalNAc appear at about 97-104ppm, while the C1 peaks of ΔUA appear at about 103.5ppm, the chemical shift of C4 is 106.8ppm, and the chemical shift of C5 About 148.5ppm.

According to the comprehensive hydrogen spectrum, carbon spectrum and correlation spectrum, dLFG-1A is mainly composed of monosaccharides, and GlcUA and GalNAc are connected to each other through β(1→3) and β(1→4) glycosidic bonds to form the glycan backbone. Constituting the main chain disaccharide structural unit. According to the H2 and H3 chemical shifts of GlcUA combined with ¹ H- ¹ H ROESY and ¹ H- ¹³ C HMBC, it can be judged that Fuc is linked to GlcUA by α(1→3) glycosidic bonds. Obviously, in dLFG-1A, the hexuronic acid at the non-reducing end is mainly ΔUA.

Table 2d LFG-1A ¹ H/ ¹³ C NMR signal assignment (δ[ppm])

Among them: GalNAc4S6S is 4,6-disulfate of GalNAc; Fuc2S4S, -3S, -4S are -2,4-disulfate, -3-sulfate and -4-sulfate of Fuc, respectively.

[Example 2]

β-elimination and depolymerization of TAG to prepare oligo-fucosylated glycosaminoglycan derivatives (dLFG) with a series of molecular weights:

2.1 Materials:

The TAG derived from plum blossom ginseng was prepared with the method described in Example 1. Other reagents are with embodiment 1.

2.2 Method

(1) Preparation of TAG quaternary ammonium salt: 5.02 g of TAG quaternary ammonium salt was prepared according to the method described in Example 1.

(2) Preparation of TAG benzyl esters with different degrees of benzyl esterification: Add 50 mL of DMF to the TAG quaternary ammonium salt obtained in step (1) and stir to dissolve, add 25 mL of benzyl chloride, and react under stirring at 35°C. And sample about 15mL at different reaction times, add 100mL of 1:9 (v/v) saturated NaCl-absolute ethanol to the solution respectively, centrifuge at 3500rpm for 20min, and obtain the precipitate with 50mL of 1:9 (v/v) saturated NaCl - Wash three times with absolute ethanol. Dissolve the precipitate with 40 mL of deionized water, and dialyze for 24 hours with a dialysis bag with a molecular weight cut-off of 3500 kD. Each dialysis retentate was TAG benzyl ester solution with different degrees of esterification, samples were freeze-dried, and the degrees of esterification were determined to be 9%, 21%, 28%, 45% and 56% respectively by ¹ H-NMR spectroscopy.

(3) TAG benzyl ester tetrabutylammonium preparation: the TAG benzyl ester solutions of different degrees of esterification obtained in step (2) are concentrated to 6mL respectively, and it is converted into hydrogen form by the exchange resin method. Under the monitoring of the conductivity meter, 0.4 M tetrabutylammonium hydroxide solution was titrated to convert all the sulfate groups and unesterified carboxyl groups into tetrabutylammonium salts (to a pH value of about 7.5-8.0), and the resulting freeze-drying gave TAG benzyl esters of different degrees of esterification. Butylammonium 1.523g, 1.518g, 1.493g, 1.490g, 1.731g.

(4) Preparation of series molecular weight β-elimination depolymerization product dLFG: TAG benzyl ester tetrabutylammonium with different degrees of esterification obtained in step (3), add DMF/CH ₂ Cl ₂ 1mL and 0.1M NaOH for every 50mg of ammonium salt Add DMF or CH ₂ Cl ₂ and freshly prepared 100mM NaOH–EtOH at a ratio of 1mL/EtOH, the resulting solution is yellow and transparent, stir and react at 25°C for 1 hr, quickly adjust the pH to neutral with 1N HCl to terminate the reaction, and add Saturated sodium chloride 2mL, absolute ethanol 20mL, centrifuge at 3500rpm for 15min, remove supernatant to obtain precipitate. 4mL H ₂ O dissolved the obtained precipitate, and the ion exchange resin method converted the product into the hydrogen form, and adjusted the pH to 7-8 with 0.1M NaOH.

(5) Purification of a series of molecular weight β-elimination depolymerization products: transfer the solution obtained in step (4) to a 1kD dialysis bag, dialyze with deionized water for 24hr, and obtain a series of molecular weight β-elimination depolymerization products after freeze-drying dLFG-1B, dLFG-1C, dLFG-1D, dLFG-1E, and dLFG-1F.

(6) Detection of dLFG-1 product: The molecular weight, -OSO ₃ ^- /-COO ^- molar ratio, and optical rotation of dLFG-1B, -1C, -1D, -1E and -1F were detected by the method described in Example 1.

2.3 Results

The test results of physical and chemical properties of dLFG-1B, dLFG-1C, dLFG-1D, dLFG-1E and dLFG-1F are shown in Table 2 below. The test results show that the dLFG series products obtained by β-elimination and depolymerization of TAG derived from plum flower ginseng have a high yield and a narrow molecular weight distribution. The test results of the conductivity method show that the sulfate group has no significant change, and the intrinsic viscosity decreases with the molecular weight .

Table 2. Monosaccharide composition and physical and chemical properties of dLFG with a series of molecular weights

[Example 3]

Preparation of β-elimination depolymerization products of FGAG from different sea cucumber sources:

3.1 Materials

Apostichopus japonicus, red-bellied sea cucumber Holothuria edulis, Brazilian ginseng Ludwigothurea grisea, jade-footed sea cucumber Holothuria leucospilota, and black milk sea cucumber Holothuria nobilis were commercially available dried body walls.

3.2 Method

(1) The dry body wall of sea cucumber imitation sea cucumber, red-bellied sea cucumber, Brazilian ginseng, yuzu sea cucumber, and black milk sea cucumber is crushed, and 300 g of the crushed matter is taken respectively, and the FGAG contained in it is extracted as described in the method (1) of Example 1, and weighed respectively. They are AJG, HEG, LGG, HLG and HNG.

(2) Take about 1 g of AJG, HEG, LGG, HLG, and HNG respectively, and prepare the β-elimination depolymerization product dLFG according to the methods (2) to (5) described in the examples, which are respectively called dAJG, dHEG, dLGG, HLG and dHNG. The basic steps of the β-elimination method depolymerization of the FGAG are shown in FIG. 12 .

3.3 Results

Step (1) The yields of separating and purifying AJG, HEG, LGG, HLG and HNG from the dried body walls of sea cucumbers, sea cucumbers, sea cucumbers, sea cucumbers, and sea cucumbers were about 1.4%, 0.9%, and 0.8%, respectively. % and 1.1%, and its weight average molecular weight is between about 50kD～80kD. Figure 7 of this specification shows the basic characteristics of AJG, LGG and HNG as FGAG compounds through ¹ H NMR spectrum: α-L-Fuc, β-D-GalNAc and β-D-GlcUA end groups and related The characteristic proton signal is clear and unambiguous.

Step (2) prepares dAJG (8.6kD), dHEG (11.5kD), dLGG (9.3kD), HLG (10.2kD) and dHNG (9.7kD) yields from AJG, HEG, LGG, HLG and HNG at about 70 %~90% range. Figure 8 of this specification shows the characteristic signal related to the non-reducing terminal ΔUA formed by β-elimination depolymerization through ^{the 1} H NMR spectrum of HEG and dHEG.

【Example 4】

dAJG terminal reduction product preparation:

4.1 Materials

dAJG, 8.6 kD, prepared as described in Example 3. Sodium borohydride, a commercially available analytical reagent.

4.2 Method

500mg dAJG and 250mg NaBH ₄ were dissolved in 20ml 0.1N NaOH solution respectively, NaBH ₄ solution was added to dAJG solution, the resulting solution was stirred overnight at room temperature, then 200mg NaBH ₄ was added and stirring was continued for 5hr. Thereafter, add 1N HCl to adjust the pH from about 10.3 to about 2.5 (destroying excess sodium borohydride), and then adjust the pH value to neutral with 1N NaOH, then add 150 ml of absolute ethanol, centrifuge to remove the supernatant, wash with 50 ml of absolute ethanol for 2 The precipitate was dissolved in 20ml of deionized water, the 3kD dialysis bag was dialyzed in deionized water overnight, and the dialysis retentate was freeze-dried to obtain end-reduced rdAJG.

4.3 Results

As a result, rdAJG386.3 mg was obtained. The results of DNS (3,5 dinitrosalicylic acid) assay showed that the terminal reduction of rdAJG was basically complete.

【Example 5】

Preparation of the terminal reductive amination product dLFG-2A of dLFG-1A:

5.1 Materials

dLFG-1A: Prepared in Example 1. Reagents such as tyramine and sodium cyanoborohydride were commercially available and of analytical grade.

5.2 Method

(1) Terminal reductive amination of dLFG-1A: 0.1 g of dLFG-1A obtained in Example 1 was dissolved in 3.5 mL of 0.2 mM phosphate buffer (pH 8.0), and excess 80 mg of tyramine and 30 mg of cyano were added during stirring Sodium borohydride, react in a constant temperature water bath at 35°C for about 72 hours. After the reaction, add 10 mL of 95% ethanol and centrifuge to obtain a precipitate. After washing twice with 30 mL of 95% ethanol, the obtained precipitate is redissolved with 35 mL of 0.1% NaCl, centrifuged to remove insoluble matter, and the supernatant is placed in a 1KD dialyzer. The bag was dialyzed with deionized water for 24 hours, and 82 mg of dLFG-2A was obtained after freeze-drying.

(2) Physicochemical and spectral detection: molecular weight and distribution detected by HPGPC; molar ratio of -OSO ₃ ^- /-COO ^- detected by conductometric method; content of acetylgalactosamine (D-GalNAc) detected by Elson-Morgon method, glucose detected by carbazole method Alkyd acid (D-GlcUA) content, ¹ HNMR methyl peak integral area to calculate D-GalNAc/L-Fuc molar ratio (same as Example 1). The NMR spectrum was detected by AVANCE AV500 superconducting NMR instrument (500MHz) of Bruker Company in Switzerland.

5.3 Results

The dLFG-2A product yield is about 82% based on the amount of dLFG-1A; the product component detection results show that D-GalNAc:D-GlcUA:L-Fuc:-OSO ₃ ^- is about 1.00:0.98:1.10:3.60, The Mw is about 8,969, and the PDI is about 1.42, which is basically consistent with the theoretical calculation result of the LGC-1A structural unit polymerization degree of about 10; ^{the 1} HNMR spectrum of dLFG-2A is shown in Figure 9. ¹ HNMR(D ₂ O,δ[ppm]):7.25(2',6'H);6.83(3',5'H);5.65,5.36,5.28(L-Fucα1H);3.38(8'H) ; 2.82 (7'H); 2.02 (D-GalNAc, CH ₃ ); 1.30-1.32 (L-Fuc, CH ₃ ). The integral ratio of benzene ring hydrogen to H4 of ΔUA is about 1:0.28, indicating that the reducing ends of the obtained products are all reductively tyrosinated.

[Example 6]

Reductive alkylation product dHEG-PMP preparation:

6.1 Materials

HEG: FGAG compound derived from red-bellied sea cucumber, prepared in the same way as step (1) of the method described in Example 3. 1-phenyl-3-methyl-5-pyrazolone (PMP), biochemical reagent, purity 99%.

6.2 Methods and results

100 mg of HEG was treated with step (3) (4) of the method in Example 1 to obtain 132 mg of tetrabutylammonium benzyl ester of HEG. Dissolve the obtained HEG benzyl ester tetrabutylammonium (50 mg/mL) in DMF, add EtONa-EtOH (final concentration 20 mM), stir and react at 50°C for 0.5 hr, add 10 ml of 0.5 mol/LPMP methanol solution, and continue stirring for 1.5 hr. Add 10ml of water, stir and cool to room temperature, neutralize the reaction solution with 1N HCl, add 20mL of saturated sodium chloride and 200mL of absolute ethanol, and centrifuge to remove the supernatant. 10mL H2O dissolved the obtained precipitate, 3kD dialysis bag was dialyzed and then freeze-dried to obtain the product dHEG-PMP102mg.

According to the calculation of the dHEG-PMP ¹ HNMR spectrum, the ends of the obtained products have been reductively alkylated.

[Example 7]

Anticoagulant activity of TAG-derived dLFG

7.1 Materials

Samples: the series of samples dLFG-1A-1F and dLFG-2A described in Examples 1, 2, and 5, and dLFG-1G with a Mw of about 3.5 kD prepared from TAG by the β-elimination method.

Reagents: enoxaparin sodium injection (LMWH, Mw3500～5500, Sanofi-Aventis); coagulation quality control plasma, activated partial thromboplastin time (APTT) assay kit, thrombin time (TT) assay reagent The kit and prothrombin time assay kit (PT-dry powder) are all produced by TECO GmbH in Germany; all other reagents are of commercially available analytical grade.

Instrument: MC-4000 Hemagglutination Analyzer (Metron, Germany).

5.2 Method

According to the actual situation of the sample, it was dissolved in deionized water to prepare a series of concentrations, and the anticoagulant activity of the dLFG series compounds was detected with the MC-4000 hemagglutination instrument according to the instructions of the APTT, PT, and TT detection kits.

5.3 Results

The results of the anticoagulation experiments of dLFG-1A-1F and dLFG-2A are shown in Table 4.

The results in Table 4 show that both dLFG-1A～1F and dLFG-2A can significantly prolong human plasma APTT, and the drug concentration required for doubling APTT (2-fold extension of time value) is lower than 9 μg/mL, which is stronger than that of the positive control The drug enoxaparin sodium (9.31 μg/mL) shows that these derivatives can effectively inhibit endogenous blood coagulation.

The experimental results also show that dLFG-1A～1F and dLFG-2A have little effect on PT and TT, and their effect activity is weaker than that of the positive control drug enoxaparin sodium, indicating that these compounds have an effect on the extrinsic coagulation process and the coagulation common pathway smaller.

Comparing the molecular weight of the dLFG series derivatives and the activity intensity of prolonging APTT with them, it can be seen that the anticoagulant activity increases with the increase of the molecular weight. Therefore, the molecular weight is one of the important factors affecting the anticoagulant activity of the series of compounds. Generally, the preferred dLFG of the present invention has a molecular weight of not less than 3,000 Da in terms of weight average molecular weight from the viewpoint of anticoagulant activity.

Table 4 series molecular weight dLFG anticoagulant activity

^a The PT value when the drug concentration is 25 μg/mL (the PT value of the blank control group is 14.4±0.2s);

^b The TT value when the drug concentration is 12.5 μg/mL (the TT of the blank control group is 16.0±0.3s).

[Embodiment 8]

Effect of dLFG on coagulation factor activity:

8.1 Materials

Sample: dLFG-1A～dLFG-1F, as described in Example 7.

FXIIa-5963(CENTERCHEM,INC)。Reagents: human quality control plasma (TECO GmbH, Germany); enoxaparin sodium injection (LMWH, Mw3500～5500, Sanofi-Aventis); heparin (Heparin, Mw～18000, Sigma); chondroitin polysulfate (OSCS, China Institute for the Control of Pharmaceutical and Biological Products); thrombin (IIa) 100NIHU/mg, thrombin detection chromogenic substrate (CS-0138) 25mg/vial, heparin cofactor II (HC-II) 100μg/vial, both From HYPHEN BioMed (France); Factor VIII (f.VIII) 200IU/branch, product of Shanghai Raas Blood Products Co., Ltd.; f.VIII detection kit, including Reagents: R1: Human Factor X; R2: Activation Reagent, human Factor IXa, containing human thrombin, calcium and synthetic phospholipids; R3: SXa-11, Chomogenic substrate, specific for Factor Xa; R4: Tris-BSA Buffer; HYPHEN BioMed (France);

FXIIa-5963 (CENTERCHEM, INC).

Instrument: BioTek-ELx808 microplate reader (USA).

8.2 Method

(1) Detection of endogenous factor X enzyme (f.Xase, Tenase) inhibitory activity: the detection method established by f.VIII detection kit combined with f.VIII reagent. After mixing 30 μL of dLFG-1A～F solutions of serial concentrations or the control solvent (Tris-BSA buffer R ₄ ) with 2.0 IU/mL f.VIII (30 μL), add the kit reagents R2 (30 μL), R ₁ ( 30 μL), after incubation at 37°C for 2 minutes, add R ₃ (30 μL), and accurately incubate at 37°C for 2 minutes to detect OD _405nm . The method of literature (Sheehan JP & Walke EK, Blood, 2006, 107:3876-3882) was used to calculate the EC ₅₀ value of f.Xase inhibition of each sample.

(2) Detection of HC-II-dependent antithrombin (f.IIa) activity: Add 30 μL of dLFG-1A～dLFG-1F solutions of serial concentrations or control solvent (Tris-HCl buffer) into 96-well ELISA plate, Add 30 μL of 1 μM HC-II, mix, and incubate at 37°C for 1 min; then add 30 μL of 10 U/mL IIa, incubate at 37°C for 1 min, add 30 μL of 4.5 mM chromogenic substrate CS-0138, mix, accurately incubate at 37°C for 2 min, and detect OD _405nm . ΔOD was calculated according to the blank control (Tris-HCl), and the EC ₅₀ value of inhibiting IIa of each sample was calculated by the method of literature (Sheehan JP & Walke EK, Blood, 2006, 107:3876-3882).

(3) Detection of the activation activity of blood coagulation factor f.XII: the test method of f.XII activity was slightly modified according to the method of reference (Hojima et al., Blood, 1984, 63:1453-1459). After adding 30 μL of dLFG-1A～dLFG-1F solutions of serial concentrations or reference solvent (20mM Tris-HCl buffer, pH7.4) into the 96-well ELISA plate, add 30 μL of 312nM human coagulation factor XII (containing 1mM NaAc- HCl and 40mM NaCl/0.02%NaN ₃ , pH5.3), mix, and incubate at 37°C for 1min; then add 30μL of 620nM prekallikrein, incubate at 37°C for 1min, add 30μL of 6mM kallikrein chromogenic substrate , mix, incubate at 37°C, detect OD _405nm at regular intervals, and calculate the change rate of OD value.

8.3 Results

See Table 5 for the effects of dLFG-1A～dLFG-1F on the activity of coagulation factors.

Figure 10 and Figure 11 respectively show the dose-effect relationship of dLFG-1E dependent HC-II antithrombin activity and inhibition of f.Xase activity.

The influence of table 5dLFG series compounds on coagulation factor activity

The data in Table 5 and accompanying drawings 4 and 5 show that dLFG-1A～dLFG-1F prepared by the present invention have strong anti-f.Xase activity, and the effective concentration (EC ₅₀ ) of inhibiting 50% of f.Xase activity is about 0.5～ 5nM. At the same time, it also has significant HC-II-dependent antithrombin activity, and its EC ₅₀ is about 5.5-40nM. Since f.Xase is the last target of the intrinsic coagulation pathway in the coagulation cascade and is the limiting step of the coagulation process, it may be the key mechanism for the potent anticoagulant activity of these compounds.

Recent research data show that selective endogenous blood coagulation factor inhibitors can effectively avoid causing bleeding tendency when producing antithrombotic activity. Therefore, the dLFG compounds of the present invention have potential antithrombotic application value.

On the other hand, f.XII activation can lead to severe adverse symptoms such as hypotension by activating prekallikrein, and serious clinical events caused by polysulfated chondroitin (OSCS) contamination have attracted attention. The research results of the present invention show that, unlike OSCS, none of the dLFG-1A-dLFG-1F of the present invention has significant f.XII activation activity under the effective anticoagulant dose.

[Example 9]

Aqueous solution of low molecular weight fucosylated glycosaminoglycan for injection

9.1 Materials

dLFG-1E obtained by the same method as in Example 2, Mw7,066Da.

9.2 Prescription

Name of raw material Dosage dLFG-IE 50g Water for Injection l000mL Co-made 1000 pieces

9.3 Preparation process

Weigh the prescribed amount of low-molecular-weight fucosylated glycosaminoglycan sodium salt and add water for injection to the full amount, stir to dissolve completely, and perform intermittent hot-pressing sterilization. Add 0.6% medicinal activated carbon and stir for 20 minutes; use a Buchner funnel and a 3.0 μm microporous membrane to decarbonize and filter to remove the heat source. Measure the content of intermediates. After passing the test, filter it with a 0.22 μm microporous membrane; fill it into controlled vials, 1 mL per bottle, monitor the filling volume during the filling process, pass the inspection, and pack the finished product.

【Example 10】

Preparation of low molecular weight fucosylated glycosaminoglycan freeze-dried powder injection:

10.1 Materials

dLFG-2A obtained by the same method as in Example 2, Mw8,969Da.

10.2 Prescription

Name of raw material Dosage dLFG-2A 50g Water for Injection 500mL Co-made 1000 pieces

10.3 Preparation process:

Weigh the prescribed amount of low-molecular-weight fucosylated glycosaminoglycan sodium salt and add water for injection to the full amount, stir to dissolve completely, and perform intermittent hot-pressing sterilization. Add 0.6% medicinal activated carbon and stir for 20 minutes; use a Buchner funnel and a 3.0 μm microporous membrane to decarbonize and filter to remove the heat source. Measure the content of intermediates. Filter with a 0.22μm microporous membrane after passing the test; divide into controlled vials, 0.5mL per bottle, half stoppered, put in a freeze-drying box, freeze-dry according to the set freeze-drying curve, press the stopper, and release Boxes, crimped caps, visually inspected and packaged to obtain finished products.

Freeze-drying process: Put the sample into the box, lower the temperature of the partition to -40°C, and keep it for 3 hours; drop the cold trap to -50°C, and start vacuuming to 300μbar. Start sublimation: heat up to -30°C at a constant rate for 1h, keep for 2h; heat up to -20°C for 2h, keep for 8h, and keep a vacuum at 200-300μbar; then dry: heat up to -5°C for 2h, keep for 2h, and keep a vacuum at 150-200μbar ; Raise the temperature to 10°C for 0.5h, keep it for 2h, and keep the vacuum at 80～100μbar;

Claims (16)

1. A low molecular weight glycosaminoglycan derivative and pharmaceutically acceptable salts thereof, wherein:

the monosaccharide of the low molecular weight glycosaminoglycan derivative comprises hexuronic acid, hexosamine, deoxyhexose and sulfate of the monosaccharide, wherein the hexuronic acid is D-glucuronic acid and delta^4,5-hexuronic acid (4-deoxy-threo-hex-4-enopyrauronic acid), the hexosamine being acetylgalactosamine (2-N-acetylamino-2-deoxy-D-galactose) or its terminal reduction product, the deoxyhexose being L-fucose;

monosaccharides and-OSO in the low molecular weight glycosaminoglycan derivative in a molar ratio₃ ^－The composition proportion range of the (1) is that the hexuronic acid, the hexosamine, the deoxyhexose and the sulfate group are =1, (1 +/-0.3) and (3.0 +/-1.0); in terms of molar ratio,. DELTA.^4,5-the percentage of hexuronic acid to total hexuronic acid is not less than 2.5%;

the weight average molecular weight Mw of the low molecular weight glycosaminoglycan derivative ranges from 3kD to 20 kD;

the polydispersity index of the low molecular weight glycosaminoglycan derivative is between 1.0 and 1.8.

2. The low molecular weight glycosaminoglycan derivatives of claim 1, wherein the low molecular weight glycosaminoglycan derivatives are a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),

in formula (I):

n is an integer having a mean value of 2 to 20;

-D-GlcUA- β 1-, is-D-glucuronic acid- β 1-yl-;

-D-GalNAc- β 1-, is-2-deoxy-2-N-acetylamino-D-galactos- β 1-yl-;

L-Fuc-alpha 1-is-L-fucose-alpha 1-radical-;

Δ UA-1-, is Δ^4,5-hexuronic acid-1-yl (4-deoxy-threo-hex-4-enopyran uronic acid-1-yl);

r independently of one another is-OH or-OSO₃ ^－；

R' is-OH, C1-C6 alkoxy, C7-C10 aryloxy;

R₁is a group of formula (II) or (III), and in said mixture, in terms of mole ratio, R₁Is of the formula (II) and R₁In the case of compounds of the formula (III) in a ratio of not less than 2:1,

wherein R, R' is as defined above;

R₂is a group of formula (IV) or (V),

wherein R, R' is as defined above;

R₃is carbonyl, hydroxyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, C6-C12 aryl, substituted or unsubstituted five-or six-membered nitrogen-containing heterocyclic group, or-NHR₄(ii) a wherein-NHR₄R in (1)₄Is substituted or unsubstituted straight chain or branched chain C1-C6 alkyl, substituted or unsubstituted C7-C12 aryl, or substituted or unsubstituted hetero atom-containing heterocyclic aryl.

3. The low molecular weight glycosaminoglycan derivatives and the pharmaceutically acceptable salts thereof according to claim 1 or 2, wherein the weight average molecular weight of the low molecular weight glycosaminoglycan derivatives is in the range of 5kD to 12 kD; the low molecular weight glycosaminoglycan derivative has a polydispersity index value of between 1.1 and 1.5.

4. The low molecular weight glycosaminoglycan derivatives of claim 1, 2 or 3, and the pharmaceutically acceptable salts thereof, wherein the low molecular weight glycosaminoglycan derivatives are β -elimination depolymerization products of fucosylated glycosaminoglycans derived from the body wall and/or the internal organs of Holothuroidea of the phylum Echinodermata, and reductive derivatization products of the reductive ends of the depolymerization products.

5. The low molecular weight glycosaminoglycan derivatives of claim 4, wherein the echinodermata holothurian species include but are not limited to:

apostichopus japonicus;

actinopyga mauritiana, radix Cynanchi Stauntonii;

actinopyga miniris, Actinopyga miliaris;

acaudina molpadioides;

bohadschia argus, Leptospira japonica;

holothuria rosea Holothuria edulis;

holothuria nobilis selenka;

holothuria leucospilota;

holothuria sinica;

sea cucumber Holothuria vagalbuda;

american ginseng Isostichopus badionotus;

brazilian ginseng Ludwigothia grisea;

stichopus chlororonotus, Stichopus japonicus selenka;

thelenota ananas;

thelenota anax (Juhua ginseng).

6. The pharmaceutically acceptable salt of low molecular weight glycosaminoglycan according to claim 1 or 2, wherein the pharmaceutically acceptable salt is an alkali metal, alkaline earth metal salt, and organic ammonium salt of the low molecular weight glycosaminoglycan derivative.

7. The pharmaceutically acceptable salt of a low molecular weight glycosaminoglycan derivative of claim 6 wherein the pharmaceutically acceptable salt is a sodium salt, a potassium salt, or a calcium salt of the low molecular weight glycosaminoglycan derivative.

8. The process for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 1, comprising the steps of:

(1) taking fucosylated glycosaminoglycan from echinoderm as a raw material, and completely or partially converting free carboxyl on contained hexuronic acid into carboxylic ester group through esterification reaction to obtain carboxylic ester;

(2) obtaining a low molecular weight glycosaminoglycan derivative by an ester group beta-elimination reaction of the carboxylate obtained in the step (1) in a non-aqueous solvent in the presence of an alkaline reagent;

(3) carrying out reduction treatment on the reducing tail end of the low molecular weight glycosaminoglycan derivative obtained in the step (2) to obtain a tail end reduced low molecular weight glycosaminoglycan derivative;

(4) converting the carboxylic ester group into a free carboxyl group by an alkaline hydrolysis method through the low molecular weight glycosaminoglycan derivative obtained in the step (2) and the step (3).

9. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the step (1) comprises:

(i) converting the fucosylated glycosaminoglycan from an echinoderm source to a quaternary ammonium salt;

(ii) (ii) reacting the quaternary ammonium salt obtained in (i) with halogenated hydrocarbon and halogenated aromatic hydrocarbon in an aprotic solvent system to generate a derivative with completely or partially esterified carboxyl.

10. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the non-aqueous solvent in the step (2) is selected from the group consisting of ethanol, methanol, dimethylformamide, dimethylsulfoxide, CH₂Cl₂、CHCl₃One or a mixed solvent thereof; the alkaline agent is selected from NaOH, KOH, C1-C4 sodium alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine or a mixture thereof.

11. The method for producing low molecular weight glycosaminoglycan derivatives of claim 8, wherein the terminal reduction treatment in the step (3) is a reduction of the sugar group at the reducing terminal to sugar alcohols, sugar ethers, sugar esters, aromatic and/or heterocyclic substituted derivatives.

12. The method for preparing low molecular weight glycosaminoglycan derivatives and pharmaceutically acceptable salts thereof according to claim 8, wherein the terminal reduction treatment in the step (3) is reductive amination reaction of a reducing terminal to form nitrogen-containing derivatives.

13. The pharmaceutical composition of low molecular weight glycosaminoglycan or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, comprising an effective anticoagulant dose of the low molecular weight glycosaminoglycan or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of an aqueous solution for injection or a lyophilized powder for injection.

15. Use of a low molecular weight glycosaminoglycan or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 for the preparation of a medicament for the treatment and/or prevention of thrombotic disorders.

16. Use of the pharmaceutical composition according to claim 13 or 14 for the preparation of a medicament for the treatment and/or prevention of thrombotic disorders.

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