WO2014153995A1 - 一种低分子量糖胺聚糖衍生物及其药物组合物和其制备方法与应用 - Google Patents
一种低分子量糖胺聚糖衍生物及其药物组合物和其制备方法与应用 Download PDFInfo
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- WO2014153995A1 WO2014153995A1 PCT/CN2013/090131 CN2013090131W WO2014153995A1 WO 2014153995 A1 WO2014153995 A1 WO 2014153995A1 CN 2013090131 W CN2013090131 W CN 2013090131W WO 2014153995 A1 WO2014153995 A1 WO 2014153995A1
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- molecular weight
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- dlfg
- weight glycosaminoglycan
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
Definitions
- the invention belongs to the technical field of medicine, and particularly relates to a derivate of low molecular weight Fucosylated Glycosaminoglycan (dLFG) having anticoagulant activity, and a preparation method thereof, comprising the dLFG or a pharmacy thereof A pharmaceutical composition of an acceptable salt, and the use of dLFG and a pharmaceutical composition thereof for the preparation of a medicament for treating a thrombotic disease.
- dLFG low molecular weight Fucosylated Glycosaminoglycan
- Thromboembolic diseases including ischemic stroke, coronary heart disease, and venous thromboembolism, are the leading causes of death in humans.
- Antithrombotic drug therapy is the basic method for clinical prevention and treatment of thrombotic diseases.
- antithrombotic drugs including fibrinolysis, anticoagulant and antiplatelet drugs have major and common defects: bleeding tendency and serious bleeding risk.
- the classic anticoagulant heparin (f.IIa/Xa inhibitor) and coumarin anticoagulant (VitK antagonist) were used in the clinical practice in the 30s and 40s of the last century. For more than 70 years, they have been deep vein thrombosis and cardiac origin. The cornerstone of stroke and postoperative anticoagulation medications, but the risk of bleeding and associated pharmacodynamic/pharmacokinetic defects severely limits their clinical utility. Heparin and coumarin drugs all inhibit the serine protease (coagulation factor) in the blood coagulation waterfall. The individual effects of the drug are different and the factors affecting the drug effect are complex. The clinical drug needs continuous monitoring.
- LMWH low molecular weight heparin
- Fucosylated glycosaminoglycan is a glycosaminoglycan analogue derived from the body wall or viscera of echinoderma sea cucumber: FGAG has a chondroitin-like backbone with a main chain of hexose aldehyde
- the disaccharide structural unit composed of acid and hexose hexose [ ⁇ 4] D-GlcUA (pl ⁇ 3) D-GalNAc (pi ⁇ ] is formed by sequential ligation; there is fucosyl side chain substitution on the main chain of FGAG, generally It is believed that this side chain fucose sulfate is linked to D-GlcUA by a (ctl ⁇ 3) glycosidic bond; both the FGAG backbone and the side chain sugar hydroxyl group may be sulfated (Yoshida et. al, Tetrahedron Lett, 1992, 33: 4959-4962; Mourao et. al
- Naturally derived FGAGs have significant anticoagulant activity (MourSo et al" Thromb Res, 2001, 102: 167-176; Mourao et. al, J Biol Chem, 1996, 271: 23973-23984), anticoagulant activity It involves multiple coagulation factor targets, among which the activity of endogenous factor X enzyme (f.Xase, Tenase) is the strongest (Sheehan & Walke, Blood, 2006, 107: 3876-3882; Buyue & Sheehan, /oo, 2009) , 114: 3092-3100). In addition to anticoagulant activity, FGAG
- Structural modification of FGAG is one of the ways to improve its potential application value, for example, to prepare its oligomerization product, the purpose of which is to reduce its platelet and surface activation activity while retaining anticoagulant activity.
- Chinese Patent Publication Nos. CN101735336A and CN101724086A disclose a process for preparing oligomeric fucosylated glycosaminoglycans by depolymerizing fucosylated glycosaminoglycans by a fourth cycle of transition metal ion catalyzed peroxide depolymerization in an aqueous medium.
- the sugar preparation results in an oligomerization product which is a potent inhibitor of the endogenous factor X enzyme, has good anticoagulant and antithrombotic activity, and has a markedly reduced bleeding tendency, and can be used for the prevention and/or treatment of thrombotic diseases.
- European Patent EP 0811635A1 and EP 0408770A1 dehydrogenate hydrogenation of the prototype FGAG to obtain depolymerization products with molecular weights ranging from 3,000 to 80,000 and 3,000 to 42,000, respectively, which can be used for intimal hyperplasia and thrombotic diseases. Prevention and treatment. These patent applications all use a peroxide depolymerization process to obtain an oligomeric product of FGAG.
- the peroxidation depolymerization method has the advantages of better maintaining the characteristic chemical structure and anticoagulant activity of FGAG while reducing the molecular weight of the polysaccharide, and the disadvantage is that the degree of product depolymerization is difficult to control, and the reaction product generally needs continuous sampling detection to confirm The point in time at which the reaction is terminated. Fucosylated glycosaminoglycan derivatives having a ⁇ 4 ' 5 unsaturated bond at the non-reducing end have not been reported so far.
- the object of the present invention is to provide a low molecular weight Fucosylated Glycosaminoglycan derivative (dLFG) having anticoagulant activity, a preparation method thereof, comprising the dLFG or pharmaceutically acceptable thereof A pharmaceutical composition of a salt, and the use of dLFG and a pharmaceutical composition thereof for the preparation of a medicament for treating a thrombotic disease.
- dLFG Fucosylated Glycosaminoglycan derivative
- the present invention provides the following technical solutions:
- 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, hexose hexose, deoxyhexose, and sulfates of these monosaccharides.
- hexuronic acid is D-glucuronic acid (D-GlcUA) and ⁇ 4 ' 5 -hexuronic acid (4-deoxy-reo-hex-4-enpyranuronic acid, AUA)
- the aminohexose is D-acetylgalactosamine (2-indole-acetylamino-2-deoxy-D-galactose, D-GalNAc) or its terminal reduction product, and the deoxyhexose is an L-rock Alginose (L-Fuc).
- the percentage of the AUA is not less than 2.5% in terms of a molar ratio.
- the molecular weight of the dLFG of the present invention can be detected and calibrated by a high-performance gel permeation chromatograph and a laser small-angle light scattering instrument (HPGPC-LALLS); a standard curve is prepared using a series of dLFGs of a predetermined molecular weight as a standard, and ultraviolet detection is used in combination.
- High molecular weight gel chromatography (HPGPC) of a detector (UVD) and/or a differential detector (RID) can routinely detect the molecular weight of the dLFG.
- the molecular weight of the dLFG of the present invention ranges from 3 kD to 20 kD in terms of weight average molecular weight (Mw). In a preferred embodiment of the present invention, the Mw of the dLFG ranges from about 5 kD to 12 kD.
- the polydispersity index (PDI) of the dLFG of the present invention is generally between 1.0 1.8, and the so-called PDI refers to the ratio of the weight average molecular weight Mw of the dLFG to the number average molecular weight Mn.
- the dLFG has a PDI value between 1.1 and 1.5.
- the low molecular weight glycosaminoglycan derivative of the present invention is a mixture of homologous glycosaminoglycan derivatives having the structure of formula (I),
- n is an integer having a mean value of about 2 to 20; among the preferred compounds of the present invention, n is an integer having a mean value of about 4 to 12.
- -D-GalNAc- ⁇ 1 - is 2-deoxy-2-indole-acetylamino-D-galactose- ⁇ 1 -yl-;
- L-Fuc- ⁇ - is -L-fucose-al-yl-;
- R is independently -H or -S0 3 _ :
- R is -OH, C1-C6 decyloxy, C7-C12 aryloxy;
- R is a non-reducing end of the homologous glycosaminoglycan derivative of the present invention, and its structure is a group represented by the formula ( ⁇ ) or (III),
- AUA-1- is a ⁇ 4 ' 5 -hexuronic acid small group (4-deoxy-t/zreo-hex-4-enpyranuronic acid-1-yl);
- R, R' are as defined above.
- the ratio of the compound of the formula (11) and the formula (III) in a molar ratio is not less than 2:1.
- the non-reducing end of the mixture of homologous glycosaminoglycan derivatives has a molar ratio of the formula (II) and the formula (III) of not less than about 4:1.
- R 2 is a group represented by formula (IV) or (V),
- R, R' are as defined above;
- R4 is a substituted or unsubstituted linear or branched C1-C6 hydrocarbon group, a substituted or unsubstituted C7-C12 aryl group, a substituted or unsubstituted hetero atom-containing heterocyclic aryl group.
- said R 3 is -CHO or -CH 2 OH.
- the dLFG of the present invention is a depolymerization product obtained by ⁇ -elimination of fucosylated glycosaminoglycan (FGAG) extracted from the body wall and/or viscera of the echinoderma sinensis animal body and the viscera
- FGAG fucosylated glycosaminoglycan
- the Echinoderm-derived FGAG generally has the following characteristics:
- the FGAG is obtained from the body wall or viscera of the echinoderms of the sea cucumber, and the extraction preparation method thereof is
- the constituent monosaccharides of the FGAG include D-glucuronic acid (GlcUA), DN-acetyl-2-amino-2-deoxygalactose (GalNAc), and L-fucose (Fuc), which are composed of a sulfate group substituent may be present on the sugar group;
- the main chain of the FGAG contains a [-4-D-GlcUA-pl -3-D-GalNAc-pl-] repeating structural unit, and the Fuc glyco group is linked to the main chain GlcUA glycosyl group in a side chain form.
- the FGAG has the structure shown in the formula (VI):
- n is an integer having a mean of about 40 to 90;
- R is independently of each other -OH or -OSCV
- R 5 is -H or D-GlcUA- ⁇ -;
- Re is -OH, -4-D-GalNAc or a sulfate thereof.
- the echinoderms of the present invention may include, but are not limited to:
- the Echinococcus serrata can include sea cucumber, jade ginseng, and plum ginseng.
- Another object of the present invention is to provide a process for the preparation of the dLFG of the present invention and a pharmaceutically acceptable salt thereof, the method of which generally comprises the steps of:
- FGAG fucoidan-derived fucosylated glycosaminoglycan
- the FGAG carboxylate derivative obtained in the step (1) is obtained by a ⁇ -elimination reaction of a carboxylate group in a nonaqueous solvent in the presence of an alkaline reagent to obtain a non-reducing terminal ⁇ 4 ' 5 -hexose a low molecular weight glycosaminoglycan derivative (dLFG) of aldehyde acid (AUA);
- dLFG low molecular weight glycosaminoglycan derivative of aldehyde acid
- Step (2) The obtained dLFG may optionally be subjected to a reduction treatment of a reducing end, thereby obtaining a terminally reduced dLFG.
- the obtained dLFG may be selected to convert a carboxylate and/or an amide group into a free carboxyl group by an alkali hydrolysis method.
- the present invention found that the naturally derived fucosylated glycosaminoglycan FGAG is relatively stable in an alkaline aqueous solution, and under the violent reaction conditions such as elevated temperature and enhanced alkalinity, FGAG may be accompanied by a ⁇ -elimination reaction.
- the present invention establishes a ⁇ -elimination method by selectively esterifying the free carboxyl group of the hexuronic acid, thereby achieving mild conditions and maintaining the characteristic structure of the glycosylated glycosaminoglycan.
- the "characteristic integrity" means that the basic chemical structure of the depolymerized product is equivalent to the molecular structure modification except that the molecular weight is lowered and the terminal glycosyl group (including the reducing end and the non-reducing terminal glycosyl group) may be present.
- Undepolymerized polysaccharides including monosaccharide composition and ratio, repeating structure unit junctions, number and type of sulfate groups.
- the preparation method of the FGAG carboxyl esterification product is:
- ion exchange method to convert FGAG into quaternary ammonium salt for example, FGAG aqueous solution cation exchange resin to convert neutral FGAG salt into IT type FGAG;
- the quaternary amine base may include, but is not limited to, tetrabutylammonium hydroxide, dodecyltrimethylammonium hydroxide, tetramethylammonium hydroxide, tetrapropylammonium hydroxide, tetraethylammonium hydroxide, benzyltri Methyl ammonium hydroxide, benzyl triethyl ammonium hydroxide, and the like.
- FGAG quaternary ammonium salt is reacted with a stoichiometric halogenated hydrocarbon in an aprotic solvent such as dimethylformamide (DMF), and the reaction product is isolated and purified to obtain a FGAG carboxyl ester derivative.
- a stoichiometric halogenated hydrocarbon in an aprotic solvent such as dimethylformamide (DMF)
- DMF dimethylformamide
- the hydrocarbon group in the halogenated hydrocarbon includes, but is not limited to, a C1-C6 linear or branched, saturated or unsaturated, substituted or unsubstituted aliphatic hydrocarbon group; a substituted or unsubstituted C7-C12 aromatic hydrocarbon group and the like.
- the carboxylic acid ester derivative of unfractionated heparin can undergo a ⁇ -elimination reaction in an aqueous alkaline solution, whereby a low molecular weight heparin (LMWH) containing AUA at the end can be obtained.
- LMWH low molecular weight heparin
- the experimental study of the present invention surprisingly found that in a similar condition such as an aqueous solution of NaOH, the FGAG carboxyl esterification product can be completely hydrolyzed to the prototype FGAG with little or no ⁇ -elimination reaction. Therefore, the present invention has focused on a nonaqueous solvent system which can carry out the ⁇ -elimination reaction of the FGAG carboxyl ester derivative.
- the nonaqueous solvent of the step (2) is optionally ethanol, methanol, dimethylformamide, dimethyl sulfoxide, CH 2 C1 2 , CHC1 3 or a mixed solvent thereof.
- the alkaline agent is optionally NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo or a mixture thereof.
- the solution for improving the solubility of the FGAG carboxyl ester derivative in a nonaqueous solvent is to further convert it into a quaternary ammonium salt, thereby realizing the solubility of the FGAG carboxyl ester derivative in a nonaqueous solvent which satisfies the need for eliminating the reaction.
- the reaction system is carried out by: dissolving a quaternary ammonium salt of a FGAG carboxy ester derivative in a suitable nonaqueous solvent such as DMF; dissolving NaOH or other suitable base catalyst in an anhydrous lower alcohol; The FGAG carboxyesterification product quaternary ammonium salt solution is mixed with the base catalyst solution, thereby obtaining a clear ⁇ -elimination reaction system.
- the alkaline agents include, but are not limited to, NaOH, KOH, sodium C1-C4 alkoxide, ethylenediamine, tri-n-butylamine, 4-dimethylaminopyridine, diazabicyclo, or mixtures thereof.
- a preferred alkaline agent of the invention is sodium ethoxide.
- a preferred method is to add a stoichiometric amount of sodium metal to a dry anhydrous ethanol solution to produce a sodium ethoxide-ethanol solution.
- the concentration of the quaternary ammonium salt of the FGAG carboxyl ester derivative is generally from 1 to 150 mg/ml, and the concentration of the alkali catalyst is generally from 0.1 to 100 mmol/L.
- the ⁇ -elimination reaction of the FGAG carboxyl esterification product can be carried out smoothly, and the reaction is generally completed by reacting at room temperature for 0.1 to 8 hr.
- the carboxylated esterified dLFG product can be obtained by neutralizing the reaction solution with an acid (such as hydrochloric acid); or, adding an appropriate amount of water to the reaction solution without changing the basic condition of the reaction solution, room temperature
- an acid such as hydrochloric acid
- the carboxylate group contained in the depolymerized dLFG derivative can be completely hydrolyzed by holding for about 0.5 to 1 hr.
- the dLFG product containing a free carboxyl group can be obtained by neutralizing the reaction solution with an acid.
- FGAG carboxy ester preparation and its ⁇ -elimination depolymerization can be used:
- FGAG carboxy ester is obtained by neutralizing FGAG neutral salt through H + cation exchange resin column, quaternary ammonium base titration to obtain FGAG quaternary ammonium salt, and then reacting with aprotic solvent, halogenated hydrocarbon at room temperature or under heating (carboxy 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 ( ⁇ -elimination) to finally obtain dLFG ( ⁇ -elimination depolymerization product of FGAG).
- the ⁇ -elimination method according to the present invention depolymerizes, and the molecular weight of the obtained product may be between about 3 kD and 20 kD.
- the esterification product of FGAG with a carboxyl esterification rate of about 10% to 60% is used, and the Mw can be obtained by depolymerization by ⁇ -elimination reaction at about 3 kD ⁇ 20kD dLFG product.
- the dLFG obtained by the ⁇ -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).
- the reducing end may optionally be terminally modified by a reduction reaction, which may include, but is not limited to, -
- R is -OH or -OS0 3 -.
- Reduction reactions, reductive amination reactions, and reductive alkylation reactions of the aldose reducing end are well known to those skilled in the art.
- a terminal glycosyl group represented by the formula (IV) and the formula (V), wherein R 3 may be a C1-C6 ⁇ may be obtained.
- reaction product dLFG of the present invention can be purified by a method known in the art (Chinese Patent Application Publication No. CN101735336A), for example, by dialysis or ultrafiltration to remove impurities such as small molecule salts, or by gel chromatography or DEAE ions. Exchange chromatography is further purified and the like.
- a dialysis membrane or an ultrafiltration membrane package having a suitable molecular weight cutoff may be selected according to the molecular weight requirement of the target dLFG, and the molecular weight cut off is preferably 1000 Da.
- the dialysis time is determined according to the specific treatment conditions and is usually not less than 6 hours.
- the dLFG product of the present invention can also be prepared by cation exchange to form a single salt form such as an alkali metal, an alkaline earth metal salt, an organic ammonium salt or the like.
- a single salt form of the dLFG is a sodium, potassium or calcium salt.
- the salt formation process of the dLFG product may be carried out by first exchanging the sample into a hydrogen form, and then neutralizing with the corresponding base to obtain a salt corresponding to dLFG; or preferably, the dynamic ion exchange salt formation method is directly exchanged on the column to form a salt, wherein A strong acid cation exchange resin is used. Resin column pretreatment, sample loading and elution can be carried out in a conventional manner. .
- the dLFG of the present invention is prepared from a echinoderma-derived FGAG as a reaction starting material by a ⁇ -elimination reaction.
- the source animal of the starting material FGAG may be selected from, but not limited to, sea cucumber, white stalk, anal ginseng, scorpion venous, sea squash, white ginseng, red Abdominal sea cucumber, black milk sea cucumber, jade foot sea cucumber, Chinese sea cucumber, sea cucumber, American meat ginseng, Brazilian ginseng, green sea cucumber, plum ginseng, giant plum ginseng.
- the source animal comprises sea cucumber, jade sea cucumber, and plum ginseng.
- the structural feature of the FGAG derived from the Echinoderm family of the echinoderms of the present invention is the presence of GlcUA, GalNAc and fucose or its sulfate which are close to an equimolar ratio (about 1:1 ⁇ 0.3).
- GlcUA, GalNAc and fucose or its sulfate which are close to an equimolar ratio (about 1:1 ⁇ 0.3).
- Differences in sea cucumber varieties and their tissue sources, or differences in extraction methods can lead to differences in the monosaccharide composition ratio of FGAG and the degree of sulfation of polysaccharides. These differences do not affect the basic structural characteristics of FGAG.
- fucosylated glycosaminoglycans of other varieties of sea cucumber-derived FGAG-compliant basic structural features can be used to obtain the dLFG derivatives of the present invention.
- the dLFG of the present invention has potent anticoagulant activity, and its multiplying human-controlled plasma-activated partial thromboplastin time (APTT) drug concentration (the drug concentration that doubles APTT) is not higher than 9 g mL.
- APTT plasma-activated partial thromboplastin time
- the present study confirmed that the dLFG has significant inhibition of endogenous factor X enzyme (f.Xase, Tenase) activity, and heparin cofactor II (HC-II)-dependent antithrombin (Ola) activity.
- f.Xase Since f.Xase is the last target of the endogenous coagulation pathway in the clotting cascade, it is the rate-limiting step for various experimental coagulation processes (Buyue & Sheehan, Blood, 2009, 114: 3092-3100); with HC-II The dermatan sulfate, which relies on antithrombin activity, has been clinically used for antithrombotic therapy. Therefore, dLFG has potential application in clinical treatment of thromboembolic diseases.
- the dLFG of the present invention has no platelet-activating activity; in addition, the present inventors have surprisingly found that the dLFG of the present invention has no activity of activating DQI compared to its naturally-derived FGAG, and does not affect the f.XII-kallikrein system, It can cause blood pressure in experimental animals to decrease.
- the non-reducing end of the dLFG of the present invention may contain a ⁇ ⁇ saccharide group, and the existence of an unsaturated double bond in ⁇ is such that it has a maximum ultraviolet absorption (UV, ⁇ ) at about 232 to 238 nm, which is based on the ultraviolet spectrophotometry.
- UV, ⁇ maximum ultraviolet absorption
- the qualitative and quantitative analysis method of the detection method is of great value, especially when the sample dLFG content is analyzed by the liquid phase gel chromatography (HGPC) method, it can be detected by the UV detector with high sensitivity, so it is especially suitable for sample quality.
- the establishment of technical methods related to the analysis of dLFG content such as control and blood concentration analysis.
- the dLFG of the present invention has an exact anticoagulant activity and thus has an antithrombotic application value.
- dLFG has good water solubility and is therefore easy to prepare into a solution preparation or a frozen product thereof.
- the polysaccharide component its oral bioavailability is limited, and therefore it is preferably prepared into a parenteral dosage form, and the preparation of the preparation can be carried out according to a technical method well known in the art.
- the dLFG of the invention has potent anticoagulant activity and can therefore be used for the prevention and treatment of different degrees of thrombotic diseases, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral venous thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc. Accordingly, the present invention can provide the use of the composition for the preparation of a medicament for the treatment and prevention of cardiovascular diseases.
- the dLFG of the invention has potent anticoagulant activity and can therefore be used for the prevention and treatment of different degrees of thrombotic diseases, such as thrombotic cardiovascular diseases, thrombotic cerebrovascular diseases, pulmonary vein thrombosis, peripheral venous thrombosis, Deep vein thrombosis, peripheral arterial thrombosis, etc. Accordingly, the present invention can provide the use of the composition for the preparation of a medicament for the treatment and prevention of cardiovascular diseases.
- the applicant first studied and established the ⁇ -elimination depolymerization method of FGAG.
- the applicant found that the original FGAG is relatively stable in an alkaline solution and is less prone to ⁇ -elimination reaction.
- the ⁇ -elimination after carboxyl esterification is an optional technical method.
- the polysaccharide structure is more complicated.
- its complex steric hindrance can affect various chemical functional groups.
- the chemical reaction properties on the other hand, a variety of chemical reaction conditions can affect the stability of the side chain fucosyl glycosidic linkages.
- the present inventors have surprisingly found that the FGAG carboxyesterification product does not undergo a ⁇ -elimination reaction in an aqueous alkaline solution like the heparin esterification product.
- the ester group of the FGAG carboxyl esterification product is easily hydrolyzed, and the ⁇ -elimination reaction hardly occurs, which is significantly different from the properties of typical glycosaminoglycans such as heparin.
- the present invention successfully obtains a fucosylated glycosaminoglycan depolymerization product having a ⁇ 4 ' 5 unsaturated bond at a non-reducing end by a ⁇ -elimination reaction in a nonaqueous solvent of a FGAG carboxyl esterification product.
- dLFG depolymerized fucosylated glycosaminoglycan derivative
- 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 ⁇ 4 ' 5 unsaturated bond, the resulting dLFG is about 232-240 nm. It has the largest UV absorption ⁇ max). 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 concentration detection.
- dLFG prepared by the method of the present invention can have significant anticoagulant activity, which prolongs the activated partial thrombin time (APTT) of the host blood-controlled blood plasma, and inhibits the endogenous factor X enzyme (f. Xase, Tenase) activity and heparin cofactor II (HC-II)-dependent anti-thrombin (anti-f.XIIa) activity are basically consistent with the activity of peroxidative depolymerization products of approximate molecular weight, indicating that it has a good potential Value.
- APTT activated partial thrombin time
- the dLFG of the present invention is superior to the peroxidative depolymerization product in that it has a specific structure of a non-reducing end, has a UV absorption of about 232-240 nm by Jonax, and the quality controllability is remarkably improved; Second, 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 factor XII (f.XII) activation activity at an anticoagulant dose, thereby preventing platelet activation and f.XII-kallikrein system activation. A series of adverse reactions caused; the low molecular weight FGAG of the present invention can further reduce the bleeding tendency compared with the equivalent antithrombotic dose of heparin drugs, and has the value of treating and/or preventing thrombotic diseases.
- f.XII platelet activation and factor XII
- Figure 1 shows the HPGPC spectra of TAG and dLFG-lA
- Figure 2 (a) is a ⁇ NMR spectrum of TAG
- Figure 2 (b) is a 1H NMR spectrum of dLFG-1 A
- Figure 3 is a 13 C NMR spectrum of TAG and dLFG-lA;
- Figure 4 is a "H- 1 !! COSY NMR spectrum of TAG and dLFG-1 A;
- Figure 5(a) shows the ROESY spectrum of the TAG
- Figure 5(b) shows the TOCSY spectrum of the TAG
- Figure 5 (c) is the ROESY spectrum of dLFG-1 A
- Figure 5 (d) is the TOCSY spectrum of dLFG-1 A
- Figure 6 is a 'H- 13 C HSQC spectrum of dLFG-1 A
- Figure 7 is a 1H NMR spectrum of AJG, LGG and HNG;
- Figure 8 is a 1H NMR spectrum of HEG and dHEG
- Figure 9 is an NMR spectrum of dLFG-2A
- Figure 10 is a dose-effect relationship of dLFG-lE-dependent anti-thrombin activity of HC-II;
- Figure 11 is a dose-effect relationship of dLFG-1E inhibiting f.Xase activity
- Figure 12 is a flow chart showing the basic steps of FGAG's ⁇ -elimination depolymerization.
- Plum ginseng (73 ⁇ 4 e / e « 0 t ⁇ « a «iw Jaeger), commercially available, visceral dry body wall; benzethonium chloride, benzyl chloride, tetrabutylammonium hydroxide ( ⁇ ), ⁇ , ⁇ - ⁇
- benzethonium chloride benzyl chloride
- tetrabutylammonium hydroxide ⁇
- ⁇ - ⁇ The reagents used for methylformamide (DMF), sodium hydroxide, sodium chloride, and ethanol are all commercially available analytical reagents.
- FGAG FGAG from 73 ⁇ 4/ertota flnfl"os, TAG extraction preparation of plum ginseng source: Take dried plum ginseng dry body wall 300g, reference method (Kariya W a/., J ⁇ ⁇ / 01 ⁇ 2 ⁇ , 1990, 265(9): 5081-5085) Preparation of TAG, yield of about 1.5%, purity 98% (HPGPC, area normalization), weight average molecular weight (Mw), 65,890 Da,
- TAG quaternary ammonium salt Take 1.2g of TAG obtained in step (1) in a beaker, add 40mL of deionized water to dissolve it; titrate with 75mg/mL benzethonium chloride solution during stirring, the solution immediately has white The precipitate was formed. After the titration was completed, the mixture was centrifuged, and the precipitate was washed three times with deionized water. The final precipitate was dried under vacuum for 24 h, and then 2.68 g of FGAG ammonium salt was obtained.
- TAG benzyl esterification The TAG quaternary ammonium salt obtained in the step (2) is placed in a round bottom flask, and the force U 27 mL DMF is dissolved, and 13.5 mL of benzyl chloride is added, and the reaction is stirred at 35 ° C for 25 hours under the protection of N 2 . . After the reaction was completed, 35 mL of saturated NaCl was added to the reaction solution, 300 mL of absolute ethanol was added, and 20 inin was centrifuged at 3500 rpm, and the supernatant was removed.
- TAG benzyl ester tetrabutyl hinge salt TAG benzyl ester obtained by water-soluble step (3), ion exchange method (Dowex/r50 W > ⁇ 8 50-100(H), 60x3 cm) is converted into hydrogen form, conductance Under the monitoring of the meter, 0.4 tetrabutylammonium hydroxide solution was titrated to completely convert the sulfated and unesterified carboxyl groups into ammonium salts. The resulting solution was freeze-dried to give a total of 1.326 g of TAG benzyl ester tetrabutylammonium salt.
- the column was passed through a hydrogen-type ion exchange resin (Dowex/r50wx8 50-100(H), 60 ⁇ 3 cm), pH adjusted to neutral with O.lM NaOH, dialysis bag in IkD, deionized water dialysis After 24 h, the ⁇ -elimination depolymerization product dLFG-lA was obtained by freeze drying to about 310 mg.
- a hydrogen-type ion exchange resin Dowex/r50wx8 50-100(H), 60 ⁇ 3 cm
- HPGPC ⁇ -effect gel chromatography
- the Agilent technologies 1200 series series chromatograph, Shodex Ohpak SB-804 HQ gel column, column temperature 35 "C; mobile phase 0.1 M sodium chloride, flow rate 0.5 mL / min; Agilent 1100 RID and UVD combined detection.
- the standard curve was drawn from a series of FGAGs with molecular weights determined, and the molecular weight and distribution were calculated by GPC.
- Intrinsic viscosity According to the Chinese Pharmacopoeia (2010 edition) two appendix VI G method, Ubum viscometer.
- Monosaccharide composition assay Elson-Morgon method for the determination of acetaminogalactose (D-GalNAc) and carbazole for the determination of glucuronic acid (D-GlcUA) content (Zhang Weijie, Sugar Complex Biochemical Research Technology (Second Edition), Zhejiang : Zhejiang University Press, 1999, 19-20); 4,5 unsaturated glucuronic acid residue ( ⁇ ⁇ ) content according to the integral of H4 in iHNMR than the integral of acetylgalactose (D-GalNAc) methyl NMR spectrum of the Bruker AVANCE AV 500 superconducting nuclear magnetic resonance instrument (500 MHz) (detection conditions, solvent D 2 0, 99.9 Atom% D (Norell); internal standard, trimethylsilyl-propionic acid (TSP-d4) ; temperature 300 K).
- D-GalNAc acetaminogalactose
- D-GlcUA
- UV detection A solution of 0.855 mg/mL dLFG was scanned at a wavelength range of 190-400 nm on Shimadzu UV-2450.
- TAG and dLFG-lA The physicochemical properties of TAG and its depolymerization product dLFG-lA and the detection results of monosaccharide composition are shown in Table I.
- TAG and dLFG-lA The physicochemical properties of TAG and its depolymerization product dLFG-lA and the detection results of monosaccharide composition are shown in Table I.
- composition ratios of hexose, hexuronic acid (UA, which is the sum of GlcUA and JA) and deoxyhexose (Fuc) of TAG and dLFG-lA remained basically stable. It can be seen from the 1H NMR spectrum described later that TAG does not contain ⁇ , and the molar ratio of AUA contained in dLFG-lA to GalNAc is about 0.18:1 (in terms of molar ratio, AUA accounts for total hexouronic acid). About 7.5%).
- the UV spectrophotometer scans from 190 nm to 400 nm.
- the maximum UV absorption of dLFG is Xmax 236 nm, which is consistent with the presence of ⁇ unsaturated bonds.
- Figure 2 of the present invention shows the 'H NMR spectrum of TAG and dLFG-lA;
- Figure 3 shows the 13 CNR spectrum of TAG and dLFG-lA;
- Figure 4 shows the COSY NMR spectrum of TAG and dLFG-1
- Figure 5(a) shows the ROESY spectrum of TAG;
- Figure 5(b) shows the TOCSY spectrum of TAG;
- Figure 5(c) shows the iH- 1 !! ROESY spectrum of dLFG-lA;
- d) shows the TOCSY spectrum of dLFG-lA;
- Figure 6 shows the 1H- 13 C HSQC spectrum of dLFG-lA.
- the spectral signal of the 1H NMR of TAG and its related spectrum can be referred to the Chinese Patent Publication No. CN 102247401 A filed by the present applicant.
- the main chain GlcUA and GalNAc end group ⁇ -hydrogen signals are located at approximately 4.4 ⁇ 4.6 ppm.
- About 1.0 -1.3 ppm and 1.9 ⁇ 2.0 ppm are the methyl proton signal peaks on the Fuc methyl and GalNAc acetyl groups, respectively.
- the sugar ring hydrogen at the sulfate-substituted position appears in the range of about 4.2 to 4.8 ppm, and the signal at about 3.6 to 4.6 ppm is the superposition of the sugar ring hydrogen in the non-sulfate group-substituted position.
- the 1H NMR spectrum of dLFG-lA showed new signal peaks at about 5.76 and 5.82 ppm, which can be assigned to the 4-position H-signal of JA according to its iH NMR correlation spectrum.
- the 1H-1HCOSY and TOCSY spectra of dLFG-lA clearly show the coupling correlation between MJA's H4, H3, H2 and HI proton signals.
- the 1H-1H ROESY spectrum showed that Fuc was linked to GlcUA and AUA by ⁇ (1,3) glycosidic bonds.
- the same type of Fuc terminal hydrogen signal attached to the AUA appears at a higher field than the terminal hydrogen signal of the Fuc connected to GlcUA (see the Fuc2S4S end in Figures 2(a) and 2(b). Base hydrogen and the Fuc2S4S terminal hydrogen signal position of the AUA).
- dLFG- ⁇ is mainly composed of monosaccharides, and GlcUA and GalNAc are linked to each other to form a glycan backbone through ⁇ (1 - 3) and ⁇ (1-4) glycosidic bonds.
- the main chain disaccharide structural unit is composed.
- GlcUA's ⁇ 2 ⁇ 3 chemical shifts combined with ROESY, 1H- 13 C HMBC, it can be judged that Fuc is linked to GlcUA by ⁇ (1 ⁇ 3) glycosidic bond.
- the non-reducing end of hexuronic acid is mainly ⁇ vide
- GalNAc4S6S is 4,6-disulfate of GalNAc
- Fuc2S4S, -3S, -4S are -2,4-disulfate, -3-sulfate and -4-sulfate of Fuc, respectively.
- TAG derived from the ginseng was prepared in the same manner as in Example 1.
- Other reagents were the same as in Example 1.
- TAG quaternary ammonium salt 5.02 g of TAG quaternary ammonium salt was prepared as described in Example 1.
- TAG benzyl ester preparation Step (1) The obtained TAG quaternary ammonium salt and 50 mL of DMF were stirred and dissolved, and 25 mL of benzyl chloride was added thereto, and the reaction was carried out under stirring at 35 ° C. Approximately 15 mL were sampled at different reaction times, and 100 mL of 1:9 (v/v) saturated NaCl-anhydrous ethanol was added to the extracted solution, and centrifuged at 3500 rpm for 20 min to obtain a precipitate of 50 mL:9 (v/). v) Wash three times with saturated NaCl-anhydrous ethanol.
- the precipitate was dissolved in 40 mL of deionized water, and the 3500 kD molecular weight cut-off dialysis bag was diafiltered for 24 hours.
- Each dialysis retentate was a solution of TAG benzyl ester with different degrees of esterification.
- the samples were lyophilized, and the degree of esterification was determined by 1H-NMR spectroscopy to be 9%, 21%, 28%, 45% and 56%, respectively.
- TAG benzyl ester tetrabutylammonium The TAG benzyl ester solution solution of different esterification degree obtained in step (2) is separately concentrated to 6 mL, and converted into a hydrogen type by an exchange resin method, and monitored by a conductivity meter. The 0.4 M tetrabutylammonium hydroxide solution was titrated, and the sulfated and unesterified carboxyl groups were all converted into tetrabutylammonium salt (to a pH of about 7.5-8.0), and the obtained lyophilized to obtain TAG benzyl esters of different degrees of esterification. Tetrabutylammonium 1.523g, 1.518g, 1.493g, 1.490g, 1.731go
- step (4) Purification of series molecular weight ⁇ -elimination depolymerization product: The solution obtained in step (4) was separately transferred to a lkD dialysis bag, dialyzed in deionized water for 24 hr, and freeze-dried to obtain a series molecular weight ⁇ -elimination depolymerization product.
- dLFG-lB, dLFG-lC, dLFG-lD, dLFG-lE and dLFG-lF The solution obtained in step (4) was separately transferred to a lkD dialysis bag, dialyzed in deionized water for 24 hr, and freeze-dried to obtain a series molecular weight ⁇ -elimination depolymerization product.
- dLFG-lB, dLFG-lC, dLFG-lD, dLFG-lE and dLFG-lF The solution obtained in step (4) was separately transferred to a l
- dLFG-1 product The molecular weights of dLFG-1B, -1C, -ID, -IE and -IF, -OS0 3 7-COO-molar ratio, and optical rotation were measured as described in Example 1.
- Apostichopus japonicus, red-faced sea cucumber Holothuria edulis, Brazilian ginseng Ludwigothurea grisea, seaweed sea cucumber Holothuria leucospilota, black sea cucumber Holothuria nobilis, are commercially available dry body walls.
- Step (1) The yields of AJG, HEG, LGG, HLG and HNG from the dried body wall of sea cucumber, red sea cucumber, Brazilian ginseng, sea cucumber sea cucumber and black milk sea cucumber were about 1.4%, 0.9%, 0.8, respectively. % and 1.1%, the weight average molecular weight is between about 50kD ⁇ 80 kD.
- Figure 7 of the present specification shows the basic characteristics of AJG, LGG and HNG as FGAG compounds by 1H NMR spectrum: aL-Fuc, ⁇ -D- GalNAc and the terminal groups on ⁇ -D-GlcUA and related characteristic proton signals Clear and clear.
- Step (2) Preparation of dAJG (8.6 kD), dHEG (ll .5 kD), dLGG (9.3 kD), HLG (10.2 kD), and dHNG (9.7 kD) from AJG, HEG, LGG, HLG, and HNG is about 70% ⁇ 90% range.
- Figure 8 of the present specification shows the non-reducing terminal AUA-related characteristic signals formed by ⁇ -elimination depolymerization by the ⁇ NMR spectrum of HEG and dHEG.
- 500 mg of dAJG and 250 mg of NaBH 4 were respectively dissolved in 20 ml of 0.1 N NaOH solution, and the NaBH 4 solution was added to the dAJG solution, and the resulting solution was stirred at room temperature overnight, and then 200 mg of NaBH 4 was further added and stirring was continued for 5 hr.
- dLFG-lA dLFG-lA
- Example 1 Reagents such as tyramine and sodium cyanoborohydride are commercially available analytical grades. 5.2 method
- Terminal reductive amination of dLFG-1A The dLFG-1A O.lg obtained in Example 1 was dissolved in 3.5 mL of 0.2 mM phosphate buffer (pH 8.0), and an excess of 80 mg of tyramine and 30 mg of cyanide were separately added with stirring. Sodium borohydride was reacted in a constant temperature water bath at 35 ° C for about 72 hr. After the reaction was completed, 10 mL of 95% ethanol was added, and the precipitate was obtained by centrifugation.
- the obtained precipitate was washed twice with 30 mL of 95% ethanol, and then the precipitate was reconstituted with 35 mL of 0.1% NaCl, centrifuged to remove insoluble matter, and the supernatant was dialyzed at 1 KD. In the bag, deionized water was dialyzed for 24 h, and lyophilized to obtain dLFG-2A 82 mg.
- HPGPC detects molecular weight and distribution
- Conductance method detects -OS0 3 7-COO_ molar ratio
- Elson-Morgon method detects acetylgalactose (D-GalNAc) content
- carbazole method detects glucose aldehyde
- D-GlcUA The acid
- 1H NMR methyl peak integrated area was calculated as the D-GalNAc/L-Fuc molar ratio (same as in Example 1).
- NMR spectra were detected by the Bruker AVANCE AV 500 superconducting nuclear magnetic resonance apparatus (500 MHz).
- the yield of dLFG-2A product was about 82% based on the amount of dLFG-lA:
- the product component test results showed that
- D-GalNAc D-GlcUA: L-Fuc: -OS0 3 - is about 1.00:0.98:1.10:3.60, Mw is about 8,969, PDI is about 1.42, this and replacement page (Article 26)
- the theoretical calculation results of the polymerization degree of LGC-1A structural unit of about 10 are basically the same; the ipiNMR spectrum of dLFG-2A is shown in Fig. 9.
- HEG FGAG-like compound derived from the red-bellied sea cucumber, prepared in the same manner as in the method (1) of the method described in Example 3.
- PMP 1-phenyl-3-methyl-5 pyrazolone
- biochemical reagent purity 99%.
- HEG100 mg treated with the method step (3) (4) of Example 1, gave 132 mg of hexanes tetrabutylammonium salt.
- the obtained HEG benzyl ester tetrabutylammonium (50 mg/mL) was dissolved in DMF, and the reaction was carried out with EtONa-EtOH (final concentration: 20 mM). After stirring at 50 ° C for 0.5 hr, 10 ml of a 0.5 mol/LPMP methanol solution was added, and the reaction was further stirred. Hr.
- Reagents enoxaparin sodium injection (LMWH, Mw 3500-5500, Sanofi-Aventis); coagulation-controlled plasma, activated partial thromboplastin time (APTT) assay kit, thrombin time ( ⁇ ) determination
- LMWH enoxaparin sodium injection
- APTT activated partial thromboplastin time
- ⁇ thrombin time determination
- the kit, prothrombin time assay kit (PT-dry powder) is produced by TECO GmbH, Germany; all other reagents are commercially available analytical grade.
- the anticoagulant activity of the dLFG series compounds was determined by the MC-4000 blood coagulation instrument according to the APTT, PT, and ⁇ detection kit instructions.
- the anticoagulant activity of the dLFG series derivatives is enhanced 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.
- 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.
- the TT value at a drug concentration of 12.5 g/mL (TT of the blank control group was 16.0 ⁇ 0.3 s).
- Reagents human-controlled plasma (TECO GmbH, Germany); enoxaparin sodium injection (LMWH, Mw 3500-5500, Sanofi-Aventis); heparin (Mpar ⁇ 8000, Sigma); polysulfate Chondroitin (OSCS, China National Institute for the Control of Pharmaceutical and Biological Products); thrombin (IIa) 100 NIHU/mg, thrombin detection chromogenic substrate (CS-0138) 25 mg/viaK heparin cofactor II (HC-II) 100 g vial , all from HYPHEN BioMed (France); Factor VIII (f.VIII) 200 IU/branch, Shanghai Lai Shi Blood Products Co., Ltd.; f.VIII test kit, reagents including Reagents: Rl: Human Factor X; R2:
- Detection of endogenous factor X enzyme (f.Xase, Tenase) inhibitory activity a detection method established by using the f.VIII detection kit in combination with the f.VIII reagent.
- Serial concentration of dLFG-lA ⁇ F solution or control solvent Tris-BSA buffer ⁇ ⁇ mixed with 2.0 IU/mL f.VIII (3 ( ⁇ L)), sequentially added kit reagent R 2 (3 ( ⁇ L), ⁇ ⁇ ), after incubation for 2 min at 37 °C, add R 3 (3 (VL), 37 'C for 2 min to detect OD 405 legs.
- Literature Sheehan JP & Walke EK, Blood, 2006, 107: 3876-3882
- HC-II-dependent antithrombin (f.IIa) activity assay serial concentration of dLFG-1 A ⁇ dLFG-l F solution or control solvent (Tris-HCl buffer) 3 ( ⁇ L added to 96 wells After the plate was labeled, add 30 ⁇ 1 ⁇ of HC-II, mix and incubate for 1 min at 37 ° C; then add 30 ⁇ 10 U / mL of IIa, incubate for 1 min at 37 ° C, add 30 ⁇ 4.5 ⁇ of the chromogenic substrate CS-0138, Mix, incubate for 2 min at 37 ,, detect OD 405 nm . Calculate AOD according to blank control (Tris-HCl), literature (Sheehan JP & Walke EK, Blood, 2006, 107: 3876-3882) method to calculate EC for inhibiting Ila of each sample 5 o value.
- Figure 10 and Figure 11 show the dose-effect relationship and inhibition of f.Xase activity of dLFG-lE-dependent anti-thrombin activity of HC-II, respectively.
- f.Xase is the last target of the endogenous coagulation pathway in the clotting cascade and is a limiting step in the coagulation process, it may be a key mechanism for the potent anticoagulant activity of these compounds.
- f.XII activation can cause severe adverse symptoms such as hypotension by activating pro-kallikrein, and serious clinical events caused by polysulfate chondroitin (OSCS) contamination are of concern.
- OSCS polysulfate chondroitin
- Example 2 The same method as in Example 2 obtained dLFG-2A, Mw 8,969 Da.
- Freeze-drying process The sample is placed in the box, the temperature of the separator is lowered to -40'C for 3 hours ; the cold trap is lowered to -50'C, and the vacuum is started to 300 bar.
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US20200254003A1 (en) * | 2017-01-10 | 2020-08-13 | Jiuzhitang Co., Ltd. | Oligosaccharide compound for inhibiting intrinsic coagulation factor x-enzyme complex, and preparation method therefor and uses thereof |
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