DEPOLYMERIZED HOLOTHURIAN GLYCOSAMINOGLYCAN COMPOSITION AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

The present invention discloses a depolymerized holothurian glycosaminoglycan composition and a preparation method and application thereof. The composition comprises one or more of depolymerized holothurian glycosaminoglycans with weight-average molecular weight between 2000 Da and 12000 Da. The preparation method of the depolymerized holothurian glycosaminoglycan composition comprises the steps of extracting and purifying holothurian glycosaminoglycan, depolymerizing the holothurian glycosaminoglycan and the like. Anti-tumor studies show that the depolymerized holothurian glycosaminoglycan composition can remarkably inhibit tube formation of human umbilical vein endothelial cells in vitro and inhibit metastasis of melanomas and breast cancer in vivo. With its excellent anti-cancer properties, depolymerized holothurian glycosaminoglycan composition can be used as pharmaceuticals, nutraceuticals and other health products.

Claims

1. (canceled)

2. A preparation method of the depolymerized holothurian glycosaminoglycan composition, characterized by comprising the following steps: (1) extracting holothurian glycosaminoglycan from holothurian and purifying the holothurian glycosaminoglycan; and (2) depolymerizing the extracted holothurian glycosaminoglycan, wherein the step of depolymerizing the extracted holothurian glycosaminoglycan in step (2) comprises conducting hydrogen peroxide depolymerization reaction for 2-48 h at the controlled temperature of 20-80 C. in an acid environment, and depolymerizing to obtain the depolymerized holothurian glycosaminoglycan composition with weight-average molecular weight smaller than 12000 Da.

3. The preparation method of the depolymerized holothurian glycosaminoglycan composition according to claim 2, characterized in that the holothurian in step (1) comprises one or more of Holothuria arenicola, Holothuria atra, Holothuria leucospilota, Holothuria scabra, Holothuria nobilis, Pearsonothuria graeffei and Actinopyga mauritiana which all belong to echinodermata.

4. The preparation method of the depolymerized holothurian glycosaminoglycan composition according to claim 2, characterized in that the step of extracting the holothurian glycosaminoglycan from the holothurian in step (1) comprises conducting grinding, enzymolysis, fractional precipitation and separation of the holothurian, so that a holothurian glycosaminoglycan crude product is obtained; the enzymes for enzymolysis include one or more of alkaline protease, neutral protease, pancreatin and papain; and precipitation is conducted by means of one or more of organic solvents methyl alcohol, ethanol, isopropyl alcohol or acetone, and separation comprises ion exchange resin separation and metal salt fractional precipitation.

5. The preparation method of the depolymerized holothurian glycosaminoglycan composition according to claim 2, characterized in that the concentration of the hydrogen peroxide is 0.1-30%; acid used can be formic acid, acetic acid, propanoic acid, hydrochloric acid or sulfuric acid; and the concentration of acid is 0.1-20%.

6. The preparation method of the depolymerized holothurian glycosaminoglycan composition according to claim 2, characterized in that the step (2) of degrading the extracted holothurian glycosaminoglycan comprises conducting fractional precipitation by means of sodium chloride with different concentrations so as to achieve desalination, and then conducting freeze drying or vacuum drying, so that the depolymerized holothurian glycosaminoglycan composition with a dispersity lower than 1.5 is obtained.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. A holothurian glycosaminoglycan composition made by the preparation method according to any one of claims 2-6.

12. A healthcare product composition, characterized by comprising the depolymerized holothurian glycosaminoglycan composition according to claim 11 and carriers acceptable in healthcare industry.

13. A pharmaceutical composition, characterized by comprising the depolymerized holothurian glycosaminoglycan composition according to claim 11 and pharmaceutically acceptable carriers.

14. An application of the depolymerized holothurian glycosaminoglycan composition according to claim 11 for preventing and treating cancer.

15. The application of the depolymerized holothurian glycosaminoglycan composition to preparation of the drugs or the healthcare products for preventing and treating cancer according to claim 14, characterized by treating cancer alone or combining with other anti-tumor drugs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is molecular weight distribution of depolymerized holothurian glycosaminoglycan (dHG).

[0025] FIG. 2 is .sup.1H-NMR of holothurian glycosaminoglycan (HG) and the depolymerized holothurian glycosaminoglycan (dHG).

[0026] FIG. 3 is HUVEC tube formation result.

[0027] FIG. 4 is a lung metastatic tumor foci count histogram from a mice breast cancer model.

[0028] FIG. 5 is a lung metastatic tumor coverage histogram from a mice breast cancer model.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0029] The present invention is further described in the following embodiments, but is not limited to these embodiments.

Embodiment 1: Extraction and Purification of Holothurian Glycosaminoglycan (HG)

[0030] 1. 9.0 L of water was added to 5 kg of dried Holothurian. After 24 hours, the Holothurian was minced. Additional 11.0 L of water was added, then pancreatin 20 g was added to the mixture. The digestion lasted 6.5 hours at 50 C. and pH 7.0-8.0. The solution is filtered and 1 kg of VPOC1074 resin was added to the above filtrate, overnight resin absorption at room temperature and pH 8.0-9.0. The resin was filtered and washed with water, then washed 3 times with 2 L of 6.5% NaCl (w/w). The desired Holothurian glycosaminoglycan was eluted 3 times with 2 L of 10.5% NaCl (w/w). The combined elution (6.0 L) was precipitated with 1.2 volume of 95% ethanol (7.2 L) at 4 C. overnight. The precipitate was centrifuged at 4000 rpm for 10 min, then dehydrated twice with 95% ethanol, dried to obtain the HG.

[0031] 2. The obtained holothurian glycosaminoglycan crude product is dissolved in 20 times of water by weight. pH is adjusted with 4M NaOH solution to 10, then hydrogen peroxide is added to reach concentration of 3% (v/v) for decolorization, and the reaction mixture is stirred for 2 h at room temperature. 1% NaCl and one volume equivalent of ethyl alcohol are added to the reaction mixture, the solid is isolated and re-dissolved in 10 times of water by weigh, and followed by precipitation with 1% NaCl and ethyl alcohol. The precipitation process is repeated twice. Solid obtained is washed twice with ethyl alcohol, and then vacuum dried at 45 C. to obtain 22.5 g of a holothurian glycosaminoglycan pure product.

Embodiment 2: Preparation of Depolymerized Holothurian Glycosaminoglycan (dHG)

[0032] 1. Preparation of dHG: 20 g of the holothurian glycosaminoglycan pure product prepared in embodiment 1 was dissolved in 160 mL of water, then 19.0 mL of acetic acid and 60.0 mL of hydrogen peroxide were added. The reaction mixture is stirred for 22 h at 60 C., then cooled in an ice bath. 4M of NaOH solution is added to adjust pH to 9.5, then 2 times by volume of ethanol is added, the solid is isolated by centrifugation to obtain the depolymerized holothurian glycosaminoglycan crude product.

[0033] The crude dHG product is dissolved with 200 mL of water, then 3% by weight of NaCl is added. The dHG is precipitated out by adding 1 volume of ethyl alcohol. The precipitation is repeated once with 2% NaCl and once with 1% NaCl. The solid obtained is washed twice with ethyl alcohol, and then vacuum dried to obtain 18.0 g of depolymerized holothurian glycosaminoglycan (dHG).

[0034] 2. Physicochemical Properties of dHG

[0035] 2.1 Molecular Weight Distribution

[0036] The depolymerized holothurian glycosaminoglycan (dHG) is subjected to HPGPC analysis. Two columns are used for molecular weight analysis TSKgel G2000SW (7.8 mm30 cm, 5 m, product number: T08542) and TSKgel G3000SW (7.8 mm30 cm, 5 m, product number: T10407-02T). The weight-average molecular weight measured for dHG is 10092 Da, and dispersity is 1.3. Refer to FIG. 1 (The peak at 40.5 min is solvent peak in FIG. 1).

[0037] 2.2 Optical rotation: Following Chinese Pharmacopoeia (2010), A WZZ-IS type automatic polarimeter with a sodium light source (.sub.589nm) is used for dHG optical rotation measurement. The sample tube is 1 dm. The optical rotation for dHG is 56.5 C. while HG is 60.5 C. Results indicated that the depolymerization process of the present invention did not change structure significantly.

[0038] 2.3 Ratio of sulfate to carboxylate: Titration method with a conductivity meter was used to measure sulfate to carboxylate ratio. The ratio of sulfate to carboxylate of dHG is 3.84. This value is close to parent HG 3.55. Result demonstrated that deploymerization process in present invention did not reduce sulfate content.

[0039] 2.4 .sup.1H-NMR

[0040] .sup.1H-NMR spectra of holothurian glycosaminoglycan (HG) and depolymerized holothurian glycosaminoglycan (dHG) are compared and they are shown in FIG. 2. No significant difference is observed other than that .sup.1H NMR peaks of HG is slightly broader because of it higher molecular weight. This demonstrated that there is no or limited structure change of depolymerized HG. 1.33 ppm is the characteristic peak of methyl group from fucose ring, and 1.92 ppm is the characteristic peak of acetyl methyl group from galactosamine. The main difference for different holothurian glycosaminoglycan is sulfation in 4-position and 6-position of galactosamine in the main chain, and also difference of sulfation profile of the fucose side chain. A single peak at 4.22 ppm indicated that galactosamine of holthuria leucospilota glycosaminoglycan has 6-position sulfation rather than 4-position sulfation. This is consistent with what was reported in the literature (Fen Huizeng etc., Acta Pharmaceutica Sinica, 1983, 18(3):203). The side chain fucose group has about 15% of 2,4-disulfate group (5.65 ppm characteristic peak), about 30% of 3,4-disulfate group (5.26 ppm and 4.90 ppm characteristic peaks) and about 20% of 4-disulfate group (5.34 ppm and 5.35 ppm characteristic peaks). Other than the above reported characteristic peaks, two other peaks at 5.10 ppm and 5.00 ppm at low field, with chemical shift values slightly lower than that of the reported in the literature, indicated that 2-position or 3-position sulfate group monosubstitution on the holothurian glycosaminoglycan.

[0041] 2.5 Anticoagulation Activity

[0042] Sheep plasma activity and chromogenic assay of anti-Xa activity of the depolymerized holothurian glycosaminoglycan (dHG) are measured and compared to that of Dalteparin (a low molecular weight heparin). The result is presented in Table 1.

TABLE-US-00001 TABLE 1 Anticoagulation activity dHG 10092 Dalteparin Anti-Xa 1.1 160 Sheep Plasma Activity 6 60

[0043] The anti-Xa activity of dHG is only 1/160 of Dalteparin. The big risk of using Dalteparin is bleeding, especially at high dose for cancer patient. Low anticoagulant activity could reduce the bleeding risk significantly. Therefore much higher dose of dHG can be used in clinical.

Embodiment 3: Acute Toxicity of the Depolymerized Holothurian Glycosaminoglycan (dHG)

[0044] 1. Materials

[0045] dHG10092 (prepared with the method according to embodiment 2 of the present invention); ICR mice.

[0046] 2. Method

[0047] ICR mice are randomly divided into two groups (ten in each group, half male and half female), and are injected with 4 g/kg and 2 g/kg of dHG10092 respectively through caudal veins.

[0048] 3. Results

[0049] For the 4 g/kg dose group, part of the mice suffer from convulsion, dyspnea, abdominal respiration, righting reflex loss, uracratia and the like after caudal vein administration. These symptoms are relieved 3 min after administration, but spontaneous activity is obviously less than that of a control group.

[0050] For the 2 g/kg dose group, no obvious abnormal symptom observed after caudal vein administration. The weight of mice in both groups reduced after administration. For the 4 g/kg dose group, the weight of mice is significantly different from that of a solvent control group (P<0.05) one day after administration. The weight of mice increased two days after administration. The weight of mice in each group has no obvious difference from that of the solvent control group after two days. During an observation period, there is neither mouse dead nor obvious abnormity in each group. Observation ended 14 days after the administration. Anatomical analysis is conducted after mice are sacrificed with CO.sub.2, and no macroscopic abnormity observed in main organs of both groups. These results demonstrated that the dHG 10092 provided by the present invention is safe up to 4 g/kg in mice.

Embodiment 4: Study on Influences of the Depolymerized Holothurian Glycosaminoglycan (dHG) on Tube Formation of HUVECs

[0051] 1. Materials

[0052] Two compounds were used in this study: depolymerized holothurian glycosaminoglycan (dHG10092) having weight-average molecular weight of 10092 Da and cabozantinib

[0053] Cell strain: primary HUVECs was purchased from Allcells company.

[0054] Reagents: endothelial cell basal culture medium (Allcells, Cat#HUVEC-004B), endothelial cell complete medium (Allcells, Cat#HUVEC-004), Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (without phonol red) (GIBCO, cat#A1413202), 0.25%. Trypsin-EDTA (GIBCO, Cat#25200), fetal calf serum (GIBCO, Cat#10099141), penicillium-streptomycete double-antibody solution (GIBCO, Cat#15140-122), and DMSO(Sigma, Cat#D2650).

[0055] 2. Method:

[0056] 2.1 Samples preparation: cabozantinib is dissolved in DMSO to obtain a 20 mM of stock solution. The stock solution is further diluted 200 times in DMSO to generate final cabozantinib stock solution. The depolymerized holothurian glycosaminoglycan dHG10092 is dissolved in 0.9% NaCl to obtain a 20 mM of stock solution. The stock solution is further diluted 50 times to obtain the 400 m final dHG stock solution. When the drugs were added to HUVEC cells, dHG stock solution was further diluted to 200 m, 100 m, 50 m, and 25 m with basal culture medium.

[0057] 2.2 Tube formation experiment: 50 L/pore Geltrex gel is added to a 96-well plate, incubated for 1 h in an incubator at 37 C., and the gel is solidified. When HUVEC cell grow to 80% confluence (cultured with standard medium. One day before the experiment, the cells were starved overnight with 2% FBS basal culture medium), cells were digested with pancreatin and collected after centrifugation. The cells were re-suspended in basal culture medium (containing 2% FBS) and counted. Cell suspension is further diluted to 210.sup.5 cells/ml with the basal culture medium (containing 2% FBS), and then drugs stock solution (dHG and cabozantinib) were added to the cell suspension with ratio of 1:1. 100 L of cell suspension is added to each well in the 96-well plate (lx 10.sup.4 pieces/well) coated with the Geltrex gel. Multi-holes inspection was performed. Photos were taken after 4 hours culture, and the number of tube formed is counted. Compound toxicity is determined by CTG method. A dose-effect diagram is made with GraphPad Prism software.

[0058] 3. Results

[0059] Results indicated that dHG10092 had remarkable inhibiting effect on HUVEC tube formation when concentration reached 50 M, and the inhibition is dose dependent. Statistic analysis of tube formation is shown in FIG. 3.

Embodiment 5: Influences of Depolymerized Holothurian Glycosaminoglycan (dHG) on Murine B16F10 Melanoma Experimental Metastasis Model

[0060] 1. Materials

[0061] 1.1 Animal and Cells

[0062] C57BL/6 mice (female, 16-20 g), provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0063] 1.2. Drugs

[0064] dHG10092 (prepared with the method according to embodiment 2) and Dalteparin (Pfizer).

[0065] 1.3. Reagents

[0066] DMEM (GiBCO company from the US), phosphate buffer (PBS), calf serum, trypsin, EDTA, formaldehyde, normal saline and sodium bicarbonate.

[0067] 1.4. Instruments

[0068] Leica inverted fluorescence microscope, precise pipettor, fully-automatic high-pressure sterilization pan, bechtop, ultra-low-temperature refrigerator, CO.sub.2 incubator, pure water filter, electronic scale, desk type electrothermal blowing dry box, refrigerator, liquid nitrogen container, centrifugal machine, pH meter and injector.

[0069] 2. Method

[0070] 2.1 Preparation of Cell Suspension

[0071] Routine culture is conducted on B16F10 tumor cells. Cell passage is conducted in cell culture bottles according to a ratio of 1:10 4-5 days before experiment, so as to avoid incomplete confluence caused by cell overgrowth, wherein each culture bottle contains about 6-810.sup.6 cells. Cells in a logarithmic phase are collected, culture solution discarded, washed with PBS. 1 mL of 0.25% pancreatin-0.02% EDTA digestive solution is added, the solution is placed in a cell incubator, and after 1-3 min, the culture bottle is slightly shaken to make the cells detached. 10 mL of DMEM is added, the cells are blown with the pipette to obtain single-cell suspension, and the single-cell suspension is transferred to a 50 mL polypropylene centrifugal tube. Cells were collected by centrifugation (1200 rpm10 min), then washed twice with PBS. The cells were counted with trypan blue, and cell concentration was adjusted to be 2.510.sup.6/ml.

[0072] 2.2 Copying of a B16F10 Experimental Tumor Metastasis Model

[0073] C57BL/6J mice were randomly divided into 3 groups based on their body weight and received a single dosed subcutaneously with vehicle saline, dHG10092 at 20 mg/kg, Dalteparin at 20 mg/kg, respectively. Then these mice were intravenously implanted with the B16F10 cell suspension (210.sup.5 cells/mice). Mice were observed daily and body weights were recorded three times per week during the experiment. At day 20, mice were euthanized Lungs were collected, weighed, photos were taken and then fixed in 10% neutral for metastatic nodule count.

[0074] 3. Results

[0075] 3.1 Weight

[0076] During the experiment, no obviously body weight change was observed.

[0077] 3.2 Lung Weight

[0078] The lung weight of the drug group and the lung weight of the saline group are significantly different. The dalteparin group inhibited the increase of lung weight significantly, and dHG10092 provided by the present invention also inhibited the increase of lung weight. The inhibition rate of dalteparin reacheed 83.6%, and the inhibition rate of dHG10092 reached 55.7%.

[0079] 3.3 Lung Tumor Node Quantity Compared with the control group (normal saline group), the tumor node quantity of the dalteparin group and the tumor node number of the dHG10092 group are significantly lower. The lung tumor node quantities of the control group (normal saline group), the dalteparin group and the dHG10092 group are 99, 17 and 34 respectively.

[0080] The results indicated that the prepared depolymerized holothurian glycosaminoglycan dHG10092 can significantly inhibit tumor metastasis when used alone at 20 mg/kg, and it has no affect on mice weight.

Embodiment 6: Influences of Depolymerized Holothurian Glycosaminoglycan (dHG) on Orthotopic 4T1-Murine Mammary Carcinoma Model

[0081] 1. Materials

[0082] 1.1 Experiment Animal and Cells

[0083] BALB/C mice (female, 6-8 weeks old, 16-20 g), provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0084] 1.2. Drugs:

[0085] dHG10092 (prepared with the method according to embodiment 2 of the present invention); Cisplatin (Qilu Pharmaceutical Co., Ltd); Dalteparin (Pfizer).

[0086] 1.3. Experiment Reagents:

[0087] DMEM (GiBCO company from the US), phosphate buffer (PBS), calf serum, trypsin, EDTA, formaldehyde, normal saline and sodium bicarbonate.

[0088] 1.4. Instruments

[0089] Leica inverted fluorescence microscope, precise pipette, fully-automatic high-pressure sterilization pan, bechtop, ultra-low-temperature refrigerator, CO.sub.2 incubator, pure water filter, electronic scale, desk type electrothermal blowing dry box, refrigerator, liquid nitrogen container, centrifugal machine, pH meter and injector.

[0090] 2. Method

[0091] 2.1 Cell Culture

[0092] 4T1 cells are cultured with RPMI-1640 culture solution, wherein the concentration of added fetal calf serum is 10%, a penicillin/streptomycin double-antibody is diluted in the culture solution based on the ratio of 1:100, and the cells are cultured in a thermostatic incubator which contains 5% CO.sub.2 and has a saturation temperature of 37 C. The cells are subjected to passage every other 2-3 days based on the ratio of 1:3-1:5.

[0093] 2.2 Condition of Mouse Pulmonary Metastasis Foci when Orthotopic Tumors are Removed

[0094] BALB/C mice are randomly divided into 7 groups, and a fourth fat pad of the breast is inoculated with 110.sup.5 4 T1 cells. Then 20 mg/kg, 40 mg/kg and 80 mg/kg dHG10092 and 4 mg/kg dalteparin are injected to each group respectively. Mice were observed daily. Body weights and tumor volumes were recorded three times per week during the experiment. Breast tumors are removed after continuous administration for 12 days, and then 4 mg/kg cisplatin is adimistrated to each group from the 13th thy, once in every four days. On the 30th day, anesthesia is conducted, the mice are sacrificed, lungs and spleens are taken out, washed and weighted. Photos were taken and lung samples were fixed in 10% neutral for metastatic nodule count and HE staining

[0095] 3. Experiment Results

[0096] 3.1 Weight

[0097] The weights of all mice treated with cisplatin (alone or in a combination) reduced, indicating that cisplatin can cause weight reduction.

[0098] 3.2 Tumor Weight

[0099] Different groups have no obvious difference in primary tumor growth, however, compared with the cisplatin group, the tumor weight of the cisplatin and dHG10092 combination group is lower.

[0100] 3.3 Lung Weight and Spleen Weight

[0101] Compared with a negative control group, the group treated with cisplatin (alone or combined with dHG10092) has much lower lung weight and spleen weight, indicating that both treatment groups can inhibit tumor metastasis and diffusion of bone marrow cell relevant to tumors.

[0102] 3.4 Lung Metastasis Quantification

[0103] Compared with the group using cisplatin alone, combination of dHG10092 and cisplatin can remarkably reduce the lung tumor node quantity. It is dose-dependent and the lung tumor node quantity of the 80 mg/kg group is much less than that of the cisplatin independent group (P<0.05), as shown in FIG. 4.

[0104] HE staining and lung tumor area quantification results indicated that all cisplatin treatment groups (using cisplatin independently or with the prepared dHG10092) can remarkably reduce the lung tumor area range, the dHG10092 provided in the present invention shows a dose-effect relationship, and the 80 mg/kg dose group has the best treatment effect. As shown in FIG. 5.

[0105] The above experiment results indicated that by combining depolymerized holothurian glycosaminoglycan dHG10092 with cisplatin, tumor metastasis can be significantly inhibited, and is dose dependent. 80 mg/kg dose group gave the best result. No adverse reaction is observed.

[0106] What is described above is merely the preferred embodiments of the present invention, and it should be noted that numerous improvements and modifications can be made by those skilled in the art without deviating from the principles of the present invention, and these improvements and modifications should also be viewed to be within the scope of the present invention.