Low-molecular-weight glycosaminoglycan derivative containing terminal 2, 5-anhydrated talose or derivative thereof

Abstract

A low-molecular-weight fucosylated glycosaminoglycan (aTFG) containing 2,5-anhydrated talose, alditol, glycosylamine or N-substituted glycosylamine monosaccharide composition thereof, preparation method thereof, pharmaceutical compositions containing the aTFG, and use thereof for preventing and/or treating thrombotic diseases are provided. The aTFG has potent anticoagulant activity targeting at intrinsic coagulation factor Xase, and inhibiting thrombogenesis, and therefore can be used as drugs for preventing and/or treating cardiovascular and cerebrovascular diseases.

Claims

1. A low-molecular-weight glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof, wherein monosaccharide compositions of the low-molecular-weight glycosaminoglycan derivative comprise hexuronic acid, hexosamine, deoxyhexamethylose and 2, 5-anhydrated talose or a reduced derivative thereof; the hexuronic acid is D--glucuronic acid, the hexosamine is 2-N-acetamino-2-deoxy-D--galactose or 2-amino-2-deoxy-D--galactose or --D-2-sulfated amino-2-deoxygalactose, the deoxyhexamethylose is L--fucose, the reduced derivative of 2, 5-anhydrated talose is 2,5-anhydrated talitol or 2,5-anhydrated talosamine or N-substituted-2,5-anhydrated talosamine; based on molar ratio, the ratio of monosaccharide component content of the low-molecular-weight glycosaminoglycan derivative is hexuronic acid:hexosamine:deoxyhexamethylose=1:(10.35):(10.3); based on molar ratio, the ratio of 2, 5-anhydrated talose and/or the reduced derivative thereof is not less than 3.0% of the total monosaccharide compositions; wherein the low-molecular-weight glycosaminoglycan derivative has a weight average molecular weight (Mw) ranging from 5,000 Daltons to 12,000 Daltons, and the low-molecular-weight glycosaminoglycan derivative has a polydispersity index of between 1.1 and 1.5; further wherein the low-molecular-weight glycosaminoglycan derivative is a mixture of the homologous glycosaminoglycan derivatives having a structure of Formula (I), ##STR00005## in Formula (I): n is an integer with an average value of 3-21; -D-GlcUA-1- is --D-glucuronic acid-1-yl; -D-GalN-1- is --D-2-acetylamino-2-deoxygalactose-1-yl or --D-2-amino-2-deoxygalactose or --D-2-sulfated amino-2-deoxygalactose; L-Fuc-1- is -L-fucose-I-yl; R.sub.1 is H or -D-2-acetylamino-2-deoxygalactose sulfate-1-yl; R is OH or OSO.sub.3.sub.; R.sub.3 is H, SO.sub.3.sub. or acetyl; R.sub.2 is a group shown in Formula (II): ##STR00006## in Formula (II), -D-GlcUA-1-, L-Fuc-1-, and R are defined as above; anTal is 2,5-anhydrated talose, the alditol, glycosylamine or N-substituted glycosylamine thereof; m is 1 or 2; R4 is optionally O, O, NH.sub.2, NHR.sub.5, wherein R.sub.5 is C1-C6 straight chain or branched alkyl groups, C7-C12 aryl.

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

3. The low-molecular-weight glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof according to claim 2, wherein the pharmaceutically acceptable salt is sodium salt, potassium salt or calcium salt of the low-molecular-weight glycosaminoglycan derivative.

4. The low-molecular-weight glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the low-molecular-weight glycosaminoglycan derivative is a deaminative depolymerization product of the fucosylated glycosaminoglycan from body wall and/or viscera of an echinoderm of the class Holothuroidea, or a derivative of the depolymerization product with reduction at the reducing terminal.

5. The low-molecular-weight glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the preparation method comprises the following steps of: Step 1: treating fucosylated glycosaminoglycan from an echinoderm with hydrazine, subjecting the hexosamine therein to partial deacetylation reaction, to obtain a partially deacetylated product of the fucosylated glycosaminoglycan; wherein in Step 1, the fucosylated glycosaminoglycan from an echinoderm refers to native fucosylated glycosaminoglycan products extracted and purified from the body wall and/or viscera of an echinoderm of the class Holothuroidea; the monosaccharide components of the fucosylated glycosaminoglycan comprise D-glucuronic acid, D-N-acetylamino-2-deoxygalactose and L-fucose; the echinoderm of the class Holothuroidea is selected from the groups consisting of Thelenota ananas Jaeger, Stichopus variegates Semper, Holothuria scabra Jaeger, Holothuria leucospilota Brandt, Holothuria edulis Lesson, Bohadschia argus Jaeger, Stichopus chloronotus Brandt, Holothuria sinica Liao, Acaudina molpadioides Semper, Pearsonothuria graeffei Semper and Holothuria nobilis Selenka; wherein in Step 1, the method of deacetylation reaction treated with hydrazine comprises adding the fucosylated glycosaminoglycan from an echinoderm into anhydrous hydrazine or hydrazine hydrate solution, reacting at the temperature of 75 C.-125 C. for 2-14 hours under stirring in the presence or absence of a catalyst; further in Step 1, the deacetylation reaction treated with hydrazine is carried out in the presence of a catalyst, the catalyst is optionally selected from the groups consisting of hydrazine sulfate, hydrazine hydrochloride, hydrochloric acid or sulfuric acid, and the catalyst in the reaction solution has a concentration of 0.5%-2.5%; Step 2: treating the partially deacetylated product of the fucosylated glycosaminoglycan obtained in Step 1 with nitrous acid, subjecting it to deamination and depolymerization, to obtain a low-molecular-weight fucosylated glycosaminoglycan with 2, 5-anhydrotalosyl as a reducing terminal; optionally, subjecting the reducing terminal of the obtained low-molecular-weight fucosylated glycosaminoglycan to reduction reaction, comprising reducing 2, 5-anhydrotalosyl into an alditol, glycosylamine or N-substituted glycosylamine; wherein in Step 2, the method of the deaminative depolymerization treated with nitrous acid comprises: in ice bath or at room temperature, adding the partially deacetylated product of the fucosylated glycosaminoglycan obtained in Step 1 into 4-6 mol/L nitrous acid solution with a pH 1-5, reacting for 5-60 minutes followed by adding an alkaline solution to adjust pH to 8 or above to terminate the reaction; and then optionally process: (1) adding 3-5 volumes of ethanol to the reaction solution, standing still, centrifuging to obtain precipitation, purifying the obtained precipitation by ultrafiltration or chromatography; (2) reducing 2, 5-anhydrotalose, the reducing terminal of the reaction product, into an alditol by sodium borohydride or sodium cyanoborohydride, and then purifying the obtained product according to the procedure of Step (1); (3) reducing 2, 5-anhydrotalose, the reducing terminal of the reaction product, into a glycosylamine or N-substituted glycosylamine through reductive amination reaction, and then purifying the obtained products according to the procedure of Step (1).

6. A pharmaceutical composition comprising an anticoagulant effective amount of the low-molecular-weight glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, wherein the dosage form of the pharmaceutical composition is water solution for injection or lyophilized powder for injection.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is .sup.1H NMR spectra of FGAG and its deacetylated products, dAFG-1 and dAFG-2;

(2) FIG. 2 is .sup.13C NMR spectra of FGAG and its deacetylated products, dAFG-1 and dAFG-2;

(3) FIG. 3 is .sup.1H NMR spectra of FGAG and its deacetylated deaminative depolymerization products aTFG-a, aTFG-b and aTFG-c.

(4) FIG. 4 is .sup.13C NMR spectra of FGAG and its deacetylated deaminative depolymerization products aTFG-a, aTFG-b and aTFG-c.

(5) FIG. 5 is spectral overlay (partial) diagram of COSY (A), NOESY (B), TOCSY (C) of FGAG and its deacetylated deaminative depolymerization products aTFG-a, aTFG-b.

(6) FIG. 6 is .sup.1H COSY spectra of FGAG (A) and its deacetylated deaminative depolymerization product aTFG-b (B).

(7) FIG. 7 is the activity of aTFG for inhibiting intrinsic factor Xase.

DETAILED DESCRIPTION OF THE INVENTION

(8) The following examples are provided to illustrate the present invention in detail, but not intended to limit the scope of the invention.

Example 1

Preparation of the Deacetylated Product of FGAG (dAFG)

(9) 1.1 Materials

(10) FGAG: fucosylated glycosaminoglycan from the body wall of Thelenota ananas was prepared according to purification method disclosed in the reference (Marine Drugs, 2013, 11, 399-417), with a molecular weight of 69,930 Daltons. Hydrazine hydrate, hydrazine sulfate, hydrazine hydrochloride, hydrochloric acid, sodium chloride, anhydrous alcohol, iodic acid, hydriodic acid, sodium hydroxide and other reagents were commercially available analytical reagents.

(11) 1.2 Methods

(12) Deacetylation Reaction:

(13) 60 mg of raw material FGAG was weighed accurately and placed in a reaction tube. Optionally, 14.5 mg of hydrazine sulfate or 12.2 mg of hydrazine hydrochloride or 0.1 mL of 2.304 mol/L HCl was added as catalyst, or not added. Then 1.45 mL of hydrazine hydrate or anhydrous hydrazine was added. Under nitrogen atmosphere, the mixture was reacted at 75-105 C. for 2 to 14 hours while stirring at 250 rpm. After completion of the reaction, the reaction solution was precipitated with 80% ethanol, centrifuged to obtain precipitation, which was dried under vacuum to obtain deacetylated intermediate product sample. The sample can be directly used for nitrous acid depolymerization, or further treated to obtain relatively pure intermediate, wherein the treatment method was: the sample was evaporated and cooled in ice bath, added dropwise with 0.25 mol/L iodic acid solution until the precipitation was not dissolved and appeared to be black suspension solution. About 5 mL of 45% hydriodic acid was dropwise added, and then 3 mol/L NaOH was added to dissolve the precipitation, until the solution became clear and transparent or light yellow solution. The solution was adjusted to neutral pH, and dialyzed using a dialysis bag with 1,000 Daltons molecular weight cutoff, and freeze-dried.

(14) Determination of the Degree of Deacetylation:

(15) About 5 mg of the above deacetylated product was accurately weighed and dissolved in about 600 ml deuteroxide (TSP-containing internal standard). BUCKER DRX-500 nuclear magnetic resonance spectrometer was used to determine the sample. Degree of deacetylation (DD) was calculated by the ratio of integral area of the two methyl protons in .sup.1H NMR spectra (methyl of acetyl amino groups in acetyl galactosamines and methyl in sulfated fucose side chains).

(16) Determination of Molecular Weight of the Product:

(17) The molecular weight of the product was determined by high performance gel permeation chromatography (HPGPC). Agilent technologies 1200 series high performance liquid chromatography, Shodex Ohpak SB-804 HQ (7.8 mm300 mm) column, temperature: 35 C., detector: differential refractive index detector (G1362A). Proper sample was accurately weighed, dissolved in 0.1 mol/L sodium chloride solution, diluted to 10 mL in a 10 mL volumetric flask, mixed well, filtered through a 40 m filter membrane. The filtrate was used as a sample solution.

(18) Preparation of Standards and Reference Solution:

(19) Series dextran standards with certain molecular weight were accurately weighed, dissolved and diluted with 0.1 mol/L sodium chloride solution to obtain 10 mg/mL solution, as narrow standard correction solution. FGAG control with known molecular weight was accurately weighed, dissolved and diluted with 0.1 mol/L sodium chloride solution to obtain 10 mg/mL solution. Each 25 L of the sample, standard, control solutions was injected into the liquid chromatographic apparatus, and the chromatograms were recorded. The data were analyzed using the special GPC software.

(20) Detection of Chemical Components:

(21) The monosaccharide components acetyl galactosamine, glucuronic acid and fucose were determined by Elson-Morgon method, m-hydroxyldiphenyl method, and cysteine-phenol method, respectively (Zhang weijie, Biochemical Research Technology of Glycoconjugate 2Ed, Zhejiang: Zhejiang University Press, 1999). The molar ratio of sulfate groups to carboxyl groups was determined by conductometric method (Zhang weijie, Biochemical Research Technology of Glycoconjugate 2Ed, Zhejiang: Zhejiang University Press, 1999, 409-410).

(22) 1.3 Results

(23) The effect of the different reaction conditions on deacetylation degree of the deacetylated product (deacetylated FGAG, dAFG) is shown in Table 1. The results showed that the addition of the catalyst can accelerate the reaction process, and the deacetylation degree of the deacetylated product is higher. Deacetylated product with different deacetylation degrees can be obtained by controlling reaction time, reaction temperature and mass concentration of hydrazine.

(24) TABLE-US-00001 TABLE 1 Experimental results of the deacetylation reaction under different conditions Methyl Methyl integral integral of of fucose Deacetyl- acetylamino side ation Factor groups chains degree (DD) Reaction 2 h 1 0.92 0.065 time 6 h 1 1.03 0.167 10 h 1 1.23 0.301 14 h 1 1.31 0.342 24 h 1 1.79 0.519 36 h 1 2.79 0.691 48 h 1 3.98 0.784 Catalyst no catalyst 1 0.98 0.123 hydrazine 1 1.25 0.310 sulfate hydrazine 1 1.28 0.330 hydrochloride hydrochloride 1 1.24 0.306 acid Reaction 60 C. 1 0.88 0.010 temperature 75 C. 1 1.00 0.134 90 C. 1 1.26 0.319 105 C. 1 2.08 0.582 Hydrazine 32% 1 0.94 0.084 concentration 64% 1 1.26 0.315 100% 1 3.04 0.704

(25) FIGS. 1 and 2 showed the .sup.1H, .sup.13C-NMR spectra of two samples prepared according to the method of this example, dAFG-1 and dAFG-2, which had different deacetylation degrees. Their deacetylation degrees were calculated based on the spectra to be 48% and 88%, respectively. The spectra data also showed that before and after deacetylation, except that partial D-GalNAc was deacetylated to produce D-GalNH.sub.2, there was no significant change in the basic structure. By analysis of monosaccharide compositions before and after deacetylation, the molar ratio of the monosaccharide compositions, (D-GlcUA):(D-GalNAc+D-GalNH.sub.2):(L-Fuc), is about 1:(10.3):(10.3). This result further suggested that except that D-GalNAc was partially deacetylated, the basic structure of the polysaccharide remained s stable.

Example 2

Preparation of aTFG by Deaminative Depolymerization of dAFG

(26) 2.1 Materials

(27) dAFG, namely partially deacetylated product of FGAG, prepared according to the method of Example 1; Reagents such as sodium nitrite, concentrated sulfuric acid, sodium borohydride, sodium carbonate, sodium hydroxide, anhydrous alcohol were commercially available analytical pure reagents.

(28) 2.2 Methods

(29) Preparation of Products:

(30) About 20 mg of deacetylated intermediate products was accurately weighed and placed in a reactor, dissolved in 1 mL of water; in ice bath or at room temperature, added with 2 mL of 5.5 mol/L nitrous acid solution (pH 4), and depolymerized for 2 to 30 minutes. After completion of the reaction, 1 mol/L sodium carbonate solution was added to adjust pH to 8-9 to terminate the reaction. 1 mL of 0.1 mol/L sodium hydroxide containing 0.25 mol/L sodium borohydride was added to reduce aldehyde groups of the product depolymerized with nitrite acid, heated at 50 C. for 40 minutes. After completion of the reaction, the reaction solution was cooled to room temperature, and added with 0.5 mol/L sulfuric acid to remove excess sodium borohydride, neutralized with 0.5 mol/L sodium hydroxide solution, and dialyzed with a 1,000 Daltons dialysis bag. The dialysis fluid in the dialysis bag was collected and lyophilized.

(31) Determination of Product:

(32) The depolymerized product was determined by BUCKER DRX-500 nuclear magnetic resonance spectrometer. The molecular weight of the depolymerized sample was determined by gel exclusion chromatography. The molar ratio of sulfate groups to carboxyl groups of the depolymerized sample was determined by conductivity method.

(33) 2.3 Results

(34) The yield of final deaminative depolymerization product was more than 90%, and the purity of the sample was more than 95%.

(35) Theoretically, only free amino groups can be eliminated by nitrous acid to cleave glycosidic bonds. Therefore, according to the deacetylation degree of the sample before nitrous acid depolymerization, the number of free amino groups can be calculated and the possible molecular weight of the depolymerization products can further be calculated theoretically. The experiment results are shown in Table 2. The molecular weight of the products obtained from raw materials with different molecular weights by nitrous acid depolymerization was substantially identical to the theoretical calculated value. This indicated that based on the deacetylation degree, final depolymerized products with theoretically calculated molecular weight can be obtained.

(36) TABLE-US-00002 TABLE 2 Experiment results of the relationship between deacetylation degree and molecular weight of products Molecular Theoretical Actual weight of molecular molecular starting Deacetylation weight of weight of material degree product product 13710 11.50% 8245 8777 13710 13.79% 6577 6751 64300 15.96% 5680 7083 64300 8.26% 10984 9702

(37) The NMR spectra of the three products aTFG-a, aTFG-b and aTFG-c prepared by this example are shown in FIGS. 3 to 6. The NMR spectra of the raw materials FGAG and intermediate products dAFG were compared. According to .sup.1H NMR, .sup.13C NMR and .sup.1H homonuclear related spectra COSY, TOCSY, NOESY and .sup.1H.sup.13C heteronuclear related spectra HQSC, HMBC, the nitrous acid depolymerization products aTFG-a, aTFG-band and aTFG-c with different deacetylation degrees (15%, 48%, 88%) were subjected to signal assignment, and the NMR signal data are shown in Table 3.

(38) TABLE-US-00003 TABLE 3 .sup.1H/.sup.13C NMR signal assignments of FGAG and its deaminative depolymerization product aTFG-b H-1 H-2 H-3 H-4 H-5 H-6 Ac C-1 C-2 C-3 C-4 C-5 C-6 Ac CH.sub.3 CH.sub.3 C0 FGAG -GalNAc 4.58 4.05 3.92 4.79 3.94 416, 2.05 102.6 54.1 79.8 79.2 76.8 70.3 25.5 177.6 4S6S 4.26 -GlcUA 4.46 3.64 3.73 3.94 3.68 / / 106.8 76.8 80.5 79.8 80.4 177.6 / / -Fuc2S4S 5.66 4.48 4.14 4.83 4.88 1.34 / 99.4 78.0 69.6 84.1 69.0 18.6 / / -Fuc4S 5.33 3.82 4.01 4.81 4.91 1.34 / 101.4 71.4 74.0 80.5 69.0 18.6 / / -Fuc3S 5.37 4.15 4.69 4.03 4.52 1.25 / 101.2 71.8 83.9 73.3 69.3 18.6 / / aTFG -GalNAc 4.54 4.02 3.92 4.77 3.95 414, 2.02 102.3 51.8 78.1 79.0 74.4 67.7 23.2 177.6 4S6S 4.24 anTal4S6S 5.05 4.02 4.58 5.02 4.50 419, / 91.6 84.4 79.1 80.5 80.4 68.2 / / 4.32 / / -GlcUA 4.44 3.59 3.66 3.89 3.72 / / 106.4 76.4 80.3 79.2 79.4 177.6 / / -Fuc2S4S 5.66 4.45 4.13 4.85 4.89 1.33 / 99.0 77.8 70.1 83.6 68.8 18.5 / / -Fuc4S 5.31 3.83 3.97 4.80 4.86 1.33 / 101.3 71.1 72.6 79.9 69.0 17.8 / / -Fuc3S 5.38 4.13 4.63 4.02 4.50 1.22 / 101.2 72.8 83.5 71.3 68.8 18.2

(39) As shown in Table 3 and FIGS. 3-6, the .sup.1H NMR spectra of the deaminative depolymerization products aTFG of FGAG were substantially similar, but the signal of H-2 of D--acetyl galactosamine (D--GalNH.sub.2) at about 3.1-3.2 ppm disappeared, which was present in deacetylated FGAG, indicating that the hexosamine having free amino groups have been reacted, but there was a new signal at about 5.0-5.1 ppm, which was from reductive terminal 2,5-anhydrated talose (anTal, terminal group and H-4). Compared with the deacetylated products, .sup.1H NMR signal of the deaminative depolymerization products was closer to that of the native FGAG.

Example 3

Preparation of Nitrous Acid Depolymerization Products of FGAG from Different Sea Cucumbers

(40) 3.1 Materials

(41) Apostichopus japonicus, Holothuria edulis, Ludwigothurea grisea, Holothuria leucospilota, Holothuria nobilis, were commercially available dry body wall.

(42) 3.2 Methods

(43) (1) Each dry body wall of Apostichopus japonicus, Holothuria edulis, Ludwigothurea grisea, Holothuria leucospilota, Holothuria nobilis was crushed. 300 g of each crushed material was extracted according to the method of Example 1(1) to obtain FGAG, designated as AJG, HEG, LGG, HLG and HNG, respectively.

(44) (2) About 1 g of each AJG, HEG, LGG, HLG and HNG was weighed and used to prepare deaminative depolymerization products aTFG according to the method of Example 1 and 2, designated as aAJG, aHEG, aLGG, aHLG and aHNG respectively.

(45) 3.3 Results

(46) The yields of AJG, HEG, LGG, HLG and HNG that were extracted and purified from Apostichopus japonicus, Holothuria edulis, Ludwigothurea grisea, Holothuria leucospilota, Holothuria nobilis were about 1.4%, 0.9%, 0.8% and 1.1%, respectively. Their weight average molecular weights were from about 50,000 Daltons to 80,000 Daltons. .sup.1H NMR spectra were used to determine the structure characteristic of AJG, HEG, LGG, HLG and HNG: anomeric protons and other protons of -L-Fuc, -D-GalNAc and -D-GlcUA were clearly determined.

(47) The yields of aAJG (8,000 Daltons), aHEG (10,500 Daltons), aLGG (7,300 Daltons), HLG (10,200 Daltons) and aHNG (8,700 Daltons) prepared from AJG, HEG, LGG, HLG and HNG were about 40%-70% respectively. .sup.1H NMR spectra were used to determine the related characteristic signals of anTal obtained by depolymerization.

Example 4

Preparation of Terminal Reductive Amination Products

(48) 4.1 Materials

(49) aTFG: prepared as described in Examples 1 and 2. Tyramine, sodium cyanoborohydride and other reagents were commercially available and analytical pure.

(50) 4.2 Methods

(51) (1) Terminal Reductive Amination:

(52) About 0.1 g of the aTFG obtained in Example 2 was dissolved in 3.5 mL of 0.2 mM phosphate buffer (pH8.0), added with 80 mg of excess Tyramine and 30 mg of sodium cyanoborohydride under stirring, reacted in constant water bath at 35 C. for about 72 hours. At the end of the reaction, 10 mL of 95% ethanol was added, centrifuged to obtain precipitation. The obtained precipitation was washed twice with 30 mL of 95% ethanol, and then the precipitation was dissolved in 35 mL of 0.1% NaCl, centrifuged to remove insoluble matters. The supernatant was placed in 1,000 Daltons dialysis bag, dialyzed with deionized water for 24 hours, and lyophilized to obtain 82 mg dLFG-2A.

(53) (2) Physicochemical and Spectral Detection of Products:

(54) Molecular weight and distribution was determined by HPGPC. The OSO.sub.3.sup./COO.sup. ratio was determined by conductivity method. The content of acetyl galactosamine (D-GalNAc) was determined by Elson-Morgon method. The content of glucuronic acid (D-GlcUA) was determined by carbazole method. D-GalNAc/L-Fuc molar ratio was calculated by .sup.1H NMR methyl peak area (the same as Example 1). NMR spectra were detected by AVANCE AV 500 superconducting nuclear magnetic resonance meter (500 MHz) (Bruker company, Switzerland).

(55) 4.3 Results

(56) The yield of the products was about 72%, calculated based on the starting material. The determination results of the product compositions showed that D-GalNAc:D-GlcUA:L-Fuc:OSO.sub.3.sup. was about 1.00:0.98:1.10:3.60, Mw was about 9,969 Daltons, and PDI was about 1.32.

(57) .sup.1HNMR (D.sub.2O, [ppm]): 7.25 (2, 6H); 6.83 (3, 5H); 5.65, 5.36, 5.28 (L-Fuc1H); 3.38 (8H); 2.82 (7H); 2.02 (D-GalNAc, CH.sub.3); 1.30-1.32 (L-Fuc, CH.sub.3). The integral of protons of benzene ring to H1 of L-Fuc showed that the reducing terminals of the obtained products were completely reductive ammination by tyrosine.

Example 5

Amino Sulfation

(58) 5.1 Materials

(59) Chlorosulfonic acid, tetrabutylammonium hydroxide, dimethyl formamide, pyridine and sodium carbonate and other reagents were commercially available analytical pure.

(60) The deacetylation sample dAFG-1 was prepared from Stichopus variegates Semper according to Example 1. The degree of deacetylation was 35%.

(61) 5.2 Methods

(62) 0.10 g of dAFG-1 was accurately weighed, dissolved in 10 mL of deionized water, adjusted to pH 7.0, reacted in water bath at 40 C. 0.16 g of Na.sub.2CO.sub.3 was added one time and 0.20 g of pyridine-chlorosulfonic acid was added within 4 h, after completion of addition, further reacted for 1 hour. At the end of the reaction, the reaction solution was placed and cooled to room temperature, adjusted to pH 7.5-8.0, ultra-filtered to remove the salt, and lyophilized. The molar ratio of sulfate group to carboxyl group was determined by conductivity method.

(63) 5.3 Results

(64) The yield of the reaction products was about 87%, calculated by weight. The the molar ratio of sulfate groups to carboxyl groups was 4.3 as determined by conductivity method. It can be seen by the comparison with the native product that the free amino groups were substantially sulfated after deaceylation.

Example 6

Study on Anticoagulant Activity of aTFG

(65) 6.1 Materials

(66) aTFG sample: A series of aTFG samples with different molecular weights were prepared according to the methods of Examples 2 and 3. The physicochemical properties of these samples are shown in Table 4.

(67) Reagents and instruments: coagulation control plasma, activated partial thromboplastin time (APTT) kit, CaCl.sub.2 were manufactured by German TECO GmbH company; other reagents were commercially available and analytical pure. MC-4000 coagulometer (MDC company, Germany).

(68) TABLE-US-00004 TABLE 4 Series of aTFG samples with different molecular weights and physicochemical properties Weight Number Molar ratio average average of sulfate molecular molecular groups to Sample weight Mw weight Mn Polydispersity carboxyl No. (Daltons) (Daltons) index groups aTFG-1 24866 13514 1.84 3.60 aTFG-2 17471 9815 1.78 3.48 aTFG-3 12567 11321 1.11 3.12 aTFG-4 11332 7358 1.54 3.24 aTFG-5 10497 6439 1.63 3.42 aTFG-6 9476 6402 1.48 3.34 aTFG-7 7000 5147 1.36 3.54 aTFG-8 6600 4748 1.39 3.34 aTFG-9 5000 4166 1.20 3.42

(69) 6.2 Methods

(70) 5 L of the sample to be tested was dissolved in Tris-HCl buffer and added in 45 L coagulation control plasma, used as test sample. Activated partial thromboplastin time (APTT) kit was used to test the coagulation time.

(71) 6.3 Results (see Table 5)

(72) TABLE-US-00005 TABLE 5 APTT of aTFG with different molecular weights Drug Correlation concentration Tested coefficient for doubling sample Curve equation (R.sup.2) APTT (g/mL) aTFG-1 y = 11.577x + 31.603 0.9939 3.40 aTFG-2 y = 9.0491x + 30.793 0.9880 3.55 aTFG-3 y = 6.134x + 37.004 0.9849 4.22 aTFG-4 y = 5.756x + 35.005 0.9971 4.85 aTFG-5 y = 6.6247x + 37.21 0.9958 4.92 aTFG-6 y = 5.3358x + 37.032 0.9978 6.14 aTFG-7 y = 4.5807x + 38.73 0.9925 6.78 aTFG-8 y = 4.0239x + 38.378 0.9874 7.81 aTFG-9 y = 2.5692x + 37.982 0.9874 12.38

(73) The results of table 5 showed that the deaminative depolymerization products of FGAG, aTFG, can significantly prolong the APTT of human plasma, and the drug concentrations for doubling APTT were all less than 12 g/ml, indicating that the derivatives can effectively inhibit the intrinsic coagulation. By comparing the molecular weights of these derivatives and the drug concentrations required for doubling APTT, it was found that the larger molecular weight related to the stronger anticoagulant activity. Molecular weight is one of the main factors that affect the anticoagulant activity. According to this regular result, considering from retaining hematological activity of FGAG, the preferred aTFG of the present invention has a molecular weight of not less than 5,000 Daltons, based on weight average molecular weight.

Example 7

Inhibitory Activity for Intrinsic Factor Xase

(74) 7.1 Materials

(75) The aTFG sample was prepared according to the method of Example 2, with a molecular weight of 8,777 Daltons.

(76) Reagents and Equipment:

(77) Factor VIII (f.VIII), 200 IU/vial, Shanghai RAAS Blood Products Co., Ltd.; f.VIII test kit, 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; manufactured by HYPHEN BioMed (France). Bio Tek-ELx 808 Microplate reader (American).

(78) 7.2 Methods

(79) Determination of the inhibitory activity for intrinsic factor Xase (anti-f.Xase): The detection method established by f.VIII detection kit in conjunction with f.VIII reagent was used. 30 l of the solution to be tested with series of concentrations or blank control solution (Tris-BSA buffer R4) was mixed with 2.0 IU/ml factor VIII (30 l), added with the kit reagents R2(30 l), R1 (30 l), incubated at 37 C. for 2 minutes; then added with R3 (30 l), incubated at 37 C. for another 2 minutes, then added with 20% acetic acid (30 l) to terminate the reaction. OD.sub.405nm was detected. DOD was calculated according to the blank control (R4). EC.sub.50 values of f.Xase inhibition of the samples were calculated according to the formula disclosed in the reference (Sheehan J. P. & Walke E. K., Blood, 2006, 107:3876-3882).

(80) 7.3 Results

(81) As shown in FIG. 7., the data showed that the aTFG had an EC.sub.50 of 22 ng/mL, indicating that it had potent anti-f.Xase activity. Since intrinsic factor Xase is the last enzymatic site in the intrinsic coagulation pathway, and is the rate limiting site of the coagulation process induced by many factors, therefore, the drugs that target at this site have the least influence on physiological coagulation and hemostasis (Blood, 2010, 116(22), 4390-4391; Blood, 2009, 114, 3092-3100).

Example 8

Freeze-Dried Products

(82) 8.1 Materials

(83) According to the methods of Examples 1 and 2, FGAG from Holothuria scabra was prepared to obtain aTFG, having a weight average molecular weight of 9,476 Daltons.

(84) 8.2 Formula:

(85) TABLE-US-00006 Name of raw materials and excipent Dosage aTFG-4 50 g Water for injection 500 mL Prepared into 1000 vials

(86) 8.3 Preparation Process:

(87) Process Procedure:

(88) The formulated aTFG was weighed, added with water for injection to full capacity, stirred to dissolve completely, and subjected to interval autoclaving sterilization. 0.6% pharmaceutical activated carbon was added and stirred for 20 minutes. A Buchner funnel and a 3.0 m micro porous filter membrane were used for decarbonization filtration to remove pyrogens. The content of the intermediate was tested. The qualified products were passed through a 0.22 m micro-porous filter membrane; filled into penicillin bottles, 0.5 mL for each bottle, monitoring the filling volume during filling; partially stoppered, and transported into the lyophilizer, lyophilized according to the predetermined freeze-drying curve; completely stoppered, withdrawn from the lyophilizer, capped, inspected to be qualified, obtained the final products.

(89) Lyophilization Procedure:

(90) The samples were placed into the lyophilizer; the temperature of shelves was dropped to 40 C., maintaining for 3 hours; the temperature of cold trap was dropped to 50 C.; then the vacuum was pumped to 300 Oar. Sublimation: the temperature was increased uniformly to 30 C. within 1 hour, maintaining for 2 hours; increased uniformly to 20 C. within 2 hours, maintaining for 8 hours; the vacuum was maintained at 200-300 Oar. Drying: the temperature was increased to 5 C. within 2 hours, maintaining for 2 hours, and the vacuum was maintained at 150-200 bar; the temperature was increased to 10 C. within 0.5 hour, maintaining for 2 hours, and the vacuum was maintained at 80-100 bar.; the temperature was increased to 40 C. within 0.5 hour, maintaining for 4 hours, and the vacuum was reduced to the lowest.