WO2015000411A1 - 一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 - Google Patents
一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 Download PDFInfo
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- WO2015000411A1 WO2015000411A1 PCT/CN2014/081472 CN2014081472W WO2015000411A1 WO 2015000411 A1 WO2015000411 A1 WO 2015000411A1 CN 2014081472 W CN2014081472 W CN 2014081472W WO 2015000411 A1 WO2015000411 A1 WO 2015000411A1
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- acid polysaccharide
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- 239000008518 lycium barbarum polysaccharide Substances 0.000 description 1
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- NAYYNDKKHOIIOD-UHFFFAOYSA-N phthalamide Chemical compound NC(=O)C1=CC=CC=C1C(N)=O NAYYNDKKHOIIOD-UHFFFAOYSA-N 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
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Classifications
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- 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
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- 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
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
Definitions
- the present invention relates to the field of medicine, and more particularly to a sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof, a process for the preparation thereof and use in the preparation of a tumor growth and/or metastasis inhibitor. Background technique
- Tumors are diseases that seriously threaten human life and health. Malignant tumors have become the first cause of death for urban residents, and the second cause of death among rural residents, with the highest mortality rate among various diseases. Tumor metastasis is one of the malignant features of tumors. Metastasis and recurrence of malignant tumors are the main causes of tumor treatment failure. Therefore, the search for anti-tumor drugs capable of inhibiting tumor growth and metastasis is currently the focus of attention.
- carbohydrates are not only an important class of structural and energy substances, but also have important biological functions. They participate in the process of mutual recognition and information transmission between cells, and are considered to be in addition to nucleic acids in organisms. Another important class of information molecules, and because they are often the key factors for cell surface signal recognition, antigen-antibody reaction, intercellular information transmission and sensation, the research on biologically active polysaccharides has received increasing attention. However, due to the complicated structure of the carbohydrates, the separation and structural identification are difficult.
- the present inventors have completed extensive and intensive research to finally complete the present invention in combination with the serious situation of the tumor onset and the difficulty of treatment.
- the present inventors reacted poly-guluronic acid with a sulfonating agent at a certain temperature for a certain period of time to obtain a sulfated derivative of guluronate oligosaccharide, and then reduced by a reducing agent to prepare a sulphuric acid.
- the present invention has been completed on the basis of esterified polygulonic acid polysaccharide (olygulonic ac id sulfa te, hereinafter referred to as "PGAS").
- sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof has obvious tumor suppressing effect
- the role of growth and metastasis, its mechanism of action can inhibit heparanase (he pa na na se) activity, inhibit C-Met enzyme activity, inhibit angiogenesis, inhibit microtubule polymerization, inhibit actin depolymerization factor, etc. related.
- the present invention provides a sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof, wherein each L-guluronic acid unit is linked by a 1,4 glycosidic bond, and the reducing end 1 is a hydroxyl group.
- the C-2 position of the sugar ring is completely sulfated.
- Another object of the present invention is to provide a process for the preparation of a sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof. It is still another object of the present invention to provide a use of a sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof for the preparation of a tumor growth and/or metastasis inhibitor.
- It is still another object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of the sulfated polygulonic acid polysaccharide of the present invention or a pharmaceutically acceptable salt thereof.
- a sulfated polygulonic acid polysaccharide having the structure of the following formula (I) or a pharmaceutically acceptable salt thereof:
- n represents an integer of 0 or 1-23, and ⁇ is S0 3 H, and R 2 is each independently H or S0 3 H, provided that the degree of sulfation is converted to sulfated polygulo
- the sugar polysaccharide has a sulfur content of 5 to 20% by weight.
- the gulonic acid polysaccharide passes through the 1,4 glycosidic bond from L-guluronic acid
- the mixture is formed by a hydroxyl group at the lower end of the reducing end, a sulfuric acid esterification at the C-2 position of the sugar ring, and a partial sulfate esterification at the C-3 position.
- n is an integer of 0 or 1-23, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22 or 23;
- n is an integer from 2 to 13, more preferably n is 3, 4, 5, 6, 7, 8, 9, or 10, and most preferably n is 4, 5, 6, 7, or 8.
- the gram content is from 7 to 15% by weight, more preferably from 9 to 13% by weight.
- the biological effect of the four sugar to the twelve sugars (especially the hexasaccharide to the hexasaccharide) and/or the content of 7 to 15% by weight (especially 9 to 13% by weight) is preferred, and it is likely They are more easily recognized and accepted by the body cells.
- the pharmaceutically acceptable salt of the acidified polygulonic acid polysaccharide may be, for example, a sodium salt, a potassium salt, a calcium salt or a magnesium salt of these compounds, and among them, a sodium salt is preferred.
- the pharmaceutically acceptable salt can be obtained by a conventional method.
- the guluronic acid polysaccharide represented by the following structural formula ( ⁇ ) is reacted with a cross-linking agent, and then reduced by a reducing agent to form a sulfated poly-gulonic acid polysaccharide represented by the general formula (I).
- m represents an integer of 0 or 1-48;
- n and ⁇ and R 2 are as defined above.
- the reaction time may be 1. 5-4. 5 hours, More preferably, it is 2-3. 5 hours, and most preferably 3 hours; preferably, the reducing agent may be sodium borohydride or sodium cyanoborohydride, Nickel-hydrogen reagent or surface-reducing agent and the like.
- a sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof for the preparation of a tumor growth and/or metastasis inhibitor.
- tumor may refer to any form of tumor including a malignant tumor, for example, the tumor may be liver cancer, gastric cancer, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, kidney cancer, bladder cancer. , prostate cancer, melanoma, brain tumors, etc.
- the sulfated polygulonic acid polysaccharide or a pharmaceutically acceptable salt thereof can be used as a tumor growth inhibitor, a tumor metastasis inhibitor, an angiogenesis inhibitor, a heparanase inhibitor,
- C-Me t enzyme inhibitor C-Me t enzyme inhibitor, microtubule polymerization inhibitor, actin depolymerase activity inhibitor, and/or actin aggregation inhibitor.
- a pharmaceutical composition comprising a therapeutically effective amount of the sulfated polygulonic acid polysaccharide of the present invention or a pharmaceutically acceptable salt thereof.
- the active ingredient in the pharmaceutical composition consists of one or more of the sulfated polygulonic acid polysaccharides of the invention or a pharmaceutically acceptable salt thereof.
- the pharmaceutical composition of the present invention may contain one or more antitumor drugs or antitumor agents in addition to the sulfated polygulonic acid polysaccharide of the present invention or a pharmaceutically acceptable salt thereof.
- the drug acts as an active ingredient.
- the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier can be used conventionally in the art.
- the pharmaceutical composition can be used as a tumor growth inhibitor.
- the pharmaceutical composition can be used as a tumor metastasis inhibitor.
- the pharmaceutical composition can be used as an angiogenesis inhibitor, a heparanase inhibitor and a C-Me t enzyme inhibitor, a microtubule polymerization inhibitor, an inhibitor of actin depolymerization factor activity, and / or actin aggregation inhibitor.
- a method of treating a tumor comprising administering to a subject in need of treatment a therapeutically effective amount of a sulfated polygulonic acid polysaccharide of the present invention or a pharmaceutically acceptable thereof .
- the term "effective amount” may mean an effective amount of the dose and period required to achieve the desired effect. This effective amount may vary depending on certain factors, such as the type of disease or condition of the disease at the time of treatment, the configuration of the particular subject organ being administered, the size of the individual patient, or the severity of the disease or condition. Those skilled in the art will be able to empirically determine the effective amount of a particular compound without undue experimentation.
- the use of the sulfated polygulonic acid polysaccharide of the present invention or a pharmaceutically acceptable salt thereof for the preparation of a tumor therapeutic drug and a tumor metastasis therapeutic drug is particularly important for solving the difficulty of the current tumor treatment lacking an effective drug.
- Figure 1 shows a column separation diagram of the components of the sulfated polygulonic acid polysaccharide of the present invention.
- Fig. 2 shows the growth inhibitory effect of the sulfated polygulonose polysaccharide of the present invention on human breast cancer MDA-MB-435 orthotopic transplantation tumor.
- Figure 3 is a graph showing the inhibitory effect of the sulfated polygulonic acid polysaccharide of the present invention on lung metastasis of human breast cancer MDA-MB-435 orthotopic transplantation tumor.
- A is a typical photo of H&E staining showing metastases on the lungs.
- B is a quantitative map of PGAS inhibition of MDA-MB-435 orthotopic transplantation lung metastasis.
- the data in the figure is expressed as the mean SD of a typical experiment. *p ⁇ 0.05, **p ⁇ 0. 01, the treatment group was compared with the control group.
- Figure 4 is a graph showing the inhibitory effect of the sulfated polygulonic acid polysaccharide of the present invention on angiogenesis of human breast cancer MDA-MB-435 orthotopic transplantation tumor.
- A is a typical photograph of CD31 staining, and the arrow points to a positive place (magnification: 200 X)
- B is PGAS for human breast cancer MDA -Quantitative map of angiogenesis inhibition of MB-435 orthotopic transplantation tumors.
- the data in the graph is expressed as the mean of one experiment ⁇ 80. **P ⁇ 0.01, the treatment group was compared with the control group.
- Figure 5 shows the sulfated polygulonic acid polysaccharide of the present invention on mouse melanoma cell B 16 F 1 () experiment Inhibition of sexual lung metastasis.
- A is a typical photograph of metastases on the lung;
- B is a quantitative map of the inhibitory effect of PGAS on experimental lung metastasis of BwF ⁇ .
- the data in the figure is expressed as the mean SD of a typical experiment. Similar results can be obtained from at least two independent real horses.
- Fig. 6 shows that the sulfated polygulonic acid polysaccharide of the present invention inhibits neovascularization of chicken embryo urinary aponeurosis (CAM).
- A is a vehicle control group
- B is a 200 g/egg PGAS group
- C is a 400 ⁇ g/egg PGAS group
- D is a 800 g/egg PGAS (magnification: 40 times).
- Fig. 7 is a graph showing the inhibition of heparanase activity by the succinic acidified polyguluronic acid polysaccharide of the present invention.
- A shows an HPLC profile of the sulfated polygulonic acid polysaccharide of the present invention inhibiting heparanase activity
- B shows the sulfated polygulonic acid polysaccharide of the present invention calculated according to the results of the A chart. The inhibition rate of heparinase activity.
- Figure 8 shows the inhibition of the cell-free system of tubulin polymerization by the sulfated polygulonic acid polysaccharide of the present invention. Where A shows time dependence and B shows dose dependency.
- Figure 9 shows the inhibitory effect of the sulfated polygulonic acid polysaccharide of the present invention on cell-free system actin depolymerization.
- Figure 10 shows that the sulfated polygulonic acid polysaccharide of the present invention inhibits the depolymerization/shearing activity of actin depolymerizing factor to actin.
- the ##P ⁇ 0. 01 Cof i l in group was compared with the control group; ** ⁇ 0. 01 The drug group was compared with the Cof i l in group.
- Fig. 11 shows the activity of the respective sugar components of the sulfated polygulonic acid polysaccharide of the present invention for inhibiting tumor metastasis.
- Olyguluronic ac id purchased from Ocean University of China, Lantai Pharmaceutical Limited liability company, the weight average molecular weight relative to dextran is 1000OODa; phthalamide, chloroluic acid, etc. are provided by Sinopharm Chemical Reagent Company, all of which are analytical reagents; dextran molecular weight standard purchased from Fluka; doxorubicin injection (Adriamycin, ADM), Zhejiang Haimen Pharmaceutical Factory, Zhewei Pharmaceutical Zhunzi (1996) No.
- NEXUS-470 intelligent infrared optical language instrument NIC0LET company product
- DPX-300 nuclear magnetic resonance wave instrument Bruker products
- gel permeation chromatography (GPC) Beijing Longzhida Co., Ltd.
- UV-2102 UV-visible spectroscopic Photometer American company Unico products.
- Fresh eggs purchased from Shanghai Shenbao Chicken Farm.
- the solution was decanted, water was added to obtain a viscous material, the pH was adjusted to 7.0 with a 1% Na 2 CO 3 solution, and ethanol was precipitated with 2 volumes of a 95% ethanol solution, and the obtained precipitate was dried at 5 ⁇ T 60 ° C to obtain an ancient a 3 ⁇ 4 acidified glucosinolate of galuronic acid polysaccharide, which is formulated into a 4 mg/ml sodium acetate solution (H7.0), and added with sodium borohydride to make 50 mM, 30-40 ° 0 ⁇ , ⁇ 30 min, the reaction was stopped in a water bath, the pH was adjusted with 0.1 M acetic acid, the unreacted sodium borohydride was released, the pH was adjusted to neutrality, and the ethanol was repeatedly precipitated and washed.
- H7.0 sodium borohydride
- the crude sulfated polygulonose (PGAS) is obtained.
- the crude PGAS was mixed into a 10% solution, precipitated with a 95% ethanol solution, and the precipitate was washed with absolute ethanol. After drying, it was mixed into a 5% solution, and the impurities were filtered through a 3 ⁇ m membrane, on a Sephadex G-10 column. Desalting was carried out on (15 ⁇ 100cm), the mobile phase was water, and the fractions were collected. The eluate was detected by 3 ⁇ 4 acid-carbazole method, combined with sugar components, concentrated under reduced pressure and desalted, and lyophilized to obtain refined Sulfated polygulonic acid polysaccharide.
- the sulfur content of the sulfated polygulonic acid polysaccharide prepared above was measured by an oxygen bottle combustion method. Take about 25mg sample, accurately weighed, according to the oxygen bottle burning method for organic destruction, select 1000ml combustion bottle, concentrated hydrogen peroxide solution 0.1 ml and water 10 ml as absorption liquid, after the smoke is completely absorbed, set the ice 5 ⁇ , ⁇ In the bath for 15 minutes, the heat is slowly boiled for 2 minutes, cooled, add ethanol-ammonium acetate buffer (pH 3.7) 50ml, ethanol 30ml, 0.1% alizarin red solution 0. 3ml as indicator liquid, with high chlorine The acid bismuth titration solution (0.
- the sugar component of the fraction obtained in the preparation of the above sulfated polygulonic acid polysaccharide was subjected to structural identification.
- the above sulfated polygulonic acid polysaccharide was diluted to a suitable concentration, and distilled water was used as a blank.
- the UV-2102 ultraviolet-visible spectrophotometer was scanned between 190 nm and 400 nm, and it was found that the fraction had no specific absorption peak in the ultraviolet region. Explain that there is no conjugated double bond structure in the structure. However, there is a non-specific absorption at 190-200.
- the PGAS nuclear magnetic resonance carbon spectrum ( 13 C-awake R) was determined using a Bruker Auance DPX-300 NMR wave instrument. As a result, it was found that the uncrossed C-2 signal peak was substantially absent in the spectrum, and there was still an uncrossed C-3 signal peak. It indicates that the hydroxylation of the hydroxyl group at the C-2 position is relatively complete, and only the hydroxyl group at the C-3 position is partially sulfated.
- the molecular weight of PGAS was determined by the GPC method.
- the molecular weight standard used was Fluka's dextran
- the color column was TSK gel2000SWXL column
- the mobile phase was an aqueous solution containing 0.2% sodium azide and 2.84% Na 2 S0 4 at a flow rate of 0.5 ml/min.
- the measurement temperature was 35 °. C
- the injection volume is 25 ⁇ 1
- the detection is performed using the Refractive Index Detector detector.
- the weight average molecular weight of PGAS relative to dextran was 2513 Da
- the results of measurement on a plurality of batches of PGAS samples showed that the weight average molecular weight relative to dextran was 1500-8500 Da.
- Example 4 Poly-gulonic acid sulfated polysaccharide (the PGAS) evaluate the efficacy embodiment inhibition of tumor growth in the MDA-MB-435 cells in the logarithmic growth phase concentration of 2.5 X 10 7 breast cancer cells / ml human (from American Type Culture Collection (ATCC, Rockville, MD, USA), inoculated in 4-5 week old female mice (BALB/cA, provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences) Second nipple fat pad. When the tumor volume grew to 100-200 mm 3 , the animals were divided into negative control group and doxorubicin injection according to the tumor volume.
- ATCC American Type Culture Collection
- BALB/cA provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences
- RTV relative tumor volume
- CRTV T RTV treatment group RTV
- C RTV negative control group RTV.
- Efficacy evaluation criteria T / C % > 60 % is invalid; T / C % 60%, and statistically processed, P ⁇ 0.05 is effective.
- the animals were sacrificed 1 week after the administration was stopped.
- the number of metastatic nodules in each lung was recorded under a dissecting microscope.
- Part of the in situ tumor tissue was frozen in liquid nitrogen for extraction of total RNA and protein.
- Part of the lung and orthotopic tumor tissue were fixed with 10% formalin fixative, and the formation of blood vessels in the tumor tissue was determined by H&E staining and immunohistochemistry.
- the PGAS 5 mg/kg treatment group showed inhibition of in situ tumor growth, but the tumor inhibition effect was not significant, and the T/C value was 67.3%; PGAS The 20 mg/kg treatment group showed a significant inhibition of the growth of orthotopic xenografts in human breast cancer MDA-MB-435 mice, with a T/C value of 37.6%.
- the positive control drug ADM was shown to significantly inhibit the growth of human breast cancer MDA-MB-435 in situ tumors, and the T/C value of the ADM 5 mg/kg treatment group was 21.8%. This indicates that PGAS can significantly inhibit the growth of human breast cancer MDA-MB-435 orthotopic tumors (Fig. 2).
- PGAS inhibition rate of human breast cancer MDA-MB-435 orthotopic transplantation lung metastasis was 50.2%, 5 mg / kg, 20 mg / kg weekly intravenous administration for 7 weeks. 88. 4%.
- the inhibition rate of lung metastasis by doxorubicin (5 mg/kg) was 89.8%. This indicates that PGAS can significantly inhibit lung metastasis of human breast cancer MDA-MB-435 orthotopic xenografts (Fig. 3).
- the effect of PGAS on angiogenesis in vivo was further evaluated by immunohistochemical staining.
- Detection of the endothelial cell-specific marker CD31 indicated that there was a large amount of small angiogenesis in the orthotopic transplantation of human breast cancer MDA-MB-435. There was no significant change in the number of small blood vessels in the PGAS 5 mg/kg treated group compared with the control group. The number of small blood vessels in the PGAS 20 mg/kg treated group was significantly lower than that in the control group, and the inhibition rate was 42.1%. .
- PGAS can significantly inhibit human milk Adenocarcinoma MDA-MB-435 orthotopically transplanted intratumoral angiogenesis (Fig. 4).
- Fresh eggs (purchased from Shanghai Shenbao Chicken Farm) were placed in an incubator Rol lX base (Lyon Electr ic Company, CA, USA) at 39 ° C and 50% humidity (air chamber end up). After 7 days of continuous incubation, first identify the survival of the chicken embryo by light, and determine the location of the urinary aponeurosis, mark it, place a small hole at the end of the egg sputum, and place the chicken embryo horizontally (the urinary membrane is facing up). Wash the earballs and gently inhale, so that the urinary membrane collapses and separates from the eggshell. Allow to stand for a while, then draw a 1cm 2 square window on the chicken embryo, cut the window with scissors, and blow off the eggshell.
- PGAS 10 with a final concentration of 200, 400, 800 g/egg ⁇ 1 was added to 0. 25 x 0. 25 x 0. 25 (length x width x height) cm 3 on a gelatin sponge, and a vehicle control was set at the same time, and then the sponge was gently placed on the urethral membrane without a large blood vessel.
- the small window was sealed with a sterilized transparent tape, and incubation was continued for 48 hours.
- the tape was uncovered and photographed (magnification: 40 X) to evaluate the inhibitory effect of PGAS on angiogenesis of chicken embryo urinary decidua. As a result, it was found (Fig. 6) that the urinary sac membrane adjacent to the PGAS sponge showed a marked decrease in vascular density and a concentration-dependent inhibition tendency. This indicates that PGAS has an effect of inhibiting angiogenesis.
- Example 7 PGAS inhibits heparanase activity assay
- a serum-free insect expression system and an affinity column purification system of heparanase were constructed by PCR amplification and gene recombination, and a high activity of 95% or more purity was obtained.
- Heparanase In 150 ⁇ l of reaction buffer (50 mM sodium acetate, pH 4.2), 0.5 ⁇ M ⁇ FITC-HS (isofluoric acid-labeled heparan sulfate) and a final concentration of 25 ng/ml were added. Heparanase was added at different concentrations of PGAS and reacted at 37 °C for 3 h.
- the relative activity of the enzyme was evaluated by the decrease in the area of the first half of the intact FITC-HS to determine the effect of PGAS on heparanase activity. It was found that PGAS inhibited heparanase activity, IC 5 , in a dose-dependent manner. At 6. 55 ng/ml, when the dose was 40 ng/ml, its activity was superior to the positive control heparin (Fig. 7).
- the enzyme reaction substrate Poly (Glu, Tyr) was coated with the enzyme labeling plate, and the ATP solution and a certain concentration of sulfated poly-gulonic acid polysaccharide and c-Met enzyme solution were added, and the reaction was shaken at 37 ° C for 1 hour. 5 ⁇ After adding a BSA 5mg / ml of T-PBS diluted PY99 antibody, shaken at 37 ° C for 0.5 hours, then add horseradish peroxidase labeled goat anti-mouse IgG, shaker at 37 ° C to continue the reaction 0.
- Example 10 inhibits actin depolymerization
- aggregation buffer 100 mMTris-HCl, 20 mM MgCl 2 , 500 mM KC1, 2 mM CaCl 2 , pH 7.5
- actin depolymerization buffer 10 mM Tris_HCl, 0.2 mM CaCl 2 , 0.2 mM ATP, pH 8.0
- concentrations of PGAS dilute it to depolymerize, set up a fluorescent microplate reader, and emit light.
- the wavelength is 360 nm
- the wavelength of the absorbed light is 410 nm
- the temperature is 37 ° C
- the mixture is read once every minute for 30 min.
- PGAS significantly inhibited the depolymerization of cell-free actin in a dose-dependent manner (IC 5 , 10.6 ⁇ ) (Fig. 9).
- Example 11 PGAS inhibition cofilin depolymerization / shear actin activity
- Sulfated polygulonic acid polysaccharide was coupled with Sepharose CL-4B to prepare sulfuric acid
- the affinity chromatography column of esterified polygulonic acid polysaccharide was used to extract and separate the binding protein of the acidified polygulonic acid polysaccharide on lung cancer cell line A549 by mass spectrometry.
- Cofilin was found to be one of the stronger proteins bound to sulfated polygulonic acid polysaccharides.
- Further studies have found that sulfated polygulonic acid polysaccharide can significantly inhibit the depolymerization/shearing activity of cofilin on actin ( Figure 10). It is indicated that the sulfated polygulonic acid polysaccharide can bind to cofilin and inhibit the depolymerization/shear actin activity of cofilin, thereby inhibiting the cell migration of tumor cells.
- Example 3 The effect of different sugar fractions of PGAS isolated in Example 3 on tumor metastasis was tested by Transwell chamber.
- Pancreatic digestion MDA-MB-435 cells were cultured in vitro for several stages, and the cells were washed three times with serum-free medium and the cells were diluted to 2 X 107 ml. 100 ⁇ l of cell dilution was added to the upper chamber of each well in the Transwell chamber, 600 ⁇ l of 10% FBS was added to the lower chamber, and 100 g/ml of different PGAS sugar components were added to the upper and lower chambers. In an incubator containing 5% C0 2 at 37. C After incubating for 12 h, the culture solution was discarded, and the cells were fixed in 90% alcohol solution for 30 min.
- a pharmaceutical composition can be prepared by mixing an effective amount of the sulfated polygulonic acid polysaccharide of the present invention with a pharmaceutically acceptable carrier by a conventional formulation means.
- the pharmaceutical composition may be a tumor therapeutic drug and a tumor metastasis therapeutic drug, or may be an angiogenesis inhibitor, a heparanase inhibitor, a C-Met enzyme inhibitor, a microtubule polymerization inhibitor, and an actin depolymerization inhibitor.
- Agent The use of the sulfated polygulonic acid polysaccharide of the present invention for the preparation of a tumor therapeutic drug and a tumor metastasis therapeutic drug is of particular importance.
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Abstract
本发明提供了一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和在制备肿瘤生长和/或转移抑制剂中的用途。本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐可用于制备肿瘤生长抑制剂、肿瘤转移抑制剂、血管生成抑制剂、乙酰肝素酶抑制剂、C-Met酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂和肌动蛋白聚集抑制剂中的任一种或多种。
Description
一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 技术领域
本发明涉及医药领域， 且更具体而言， 涉及一种硫酸酯化聚古洛糖酸多 糖或其可药用盐及其制备方法和在制备肿瘤生长和 /或转移抑制剂中的用途。 背景技术
肿瘤是严重威胁人类生命和健康的疾病。 恶性肿瘤已成为城市居民的第 一死因， 农村居民的第二死因， 其死亡率居各种疾病的首位。 肿瘤转移是肿 瘤的恶性特征之一，恶性肿瘤的转移和复发是导致肿瘤治疗失败的主要原因。 因此， 寻找能够抑制肿瘤生长和转移的抗肿瘤药物是目前人们关注的焦点。
近年的研究发现， 糖类物质不仅是一类重要的结构物质和能量物质， 而 且还具有重要的生物学功能， 其参与细胞间的相互识别及信息传递过程， 被 认为是生物体内除核酸以外的又一类重要的信息分子， 而且由于它们常是细 胞表面信号识别、抗原抗体反应、 细胞间信息传递和感受的关键因子， 因此， 具有生物活性的活性多糖的研究日益受到重视。 但由于糖类物质结构复杂， 分离及结构鉴定困难， 到目前为止， 只有云芝多糖、 猪苓多糖、 香菇多糖、 裂褶多糖、 茯苓多糖等用于临床， 本领域中需要更多具有生物活性的多糖。 发明内容
针对糖类物质结构解析及制备困难， 及天然糖活性往往不理想的问题， 结合肿瘤发病形势严峻及治疗困难的现实， 本发明人经过广泛深入的研究， 最终完成本发明。 本发明人将多聚古洛糖醛酸在一定温度下与磺化剂反应一 定时间， 得到古洛糖醛酸寡糖的硫酸酯化衍生物， 再加入还原剂进行还原， 制备了石克酸酯化聚古洛糖酸多糖 ( olygulonic ac id sulfa te , 以下简称 "PGAS" ), 在此基础上完成了本发明。
因此， 本发明的一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其可药 用盐。 本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐具有明显的抑制肿瘤
生长和转移的作用， 其作用机制同其能够抑制乙酰肝素酶（ he pa r a na s e ) 活 性、 抑制 C-Met酶活性、 抑制血管生成、 抑制微管聚合、 抑制肌动蛋白解聚 因子等有关。 优选地， 本发明提供一种硫酸酯化聚古洛糖酸多糖或其可药用 盐，其中各个 L-古洛糖醛酸单元通过 1, 4糖苷键连接，其还原端 1位为羟基， 糖环 C-2位完全硫酸酯化。
本发明的另一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其可药用盐 的制备方法。 本发明的又一个目的是提供一种硫酸酯化聚古洛糖酸多糖或其 可药用盐在制备肿瘤生长和 /或转移抑制剂中的用途。
本发明的还一个目的是提供一种药物组合物， 其包含治疗有效量的本发 明的硫酸酯化聚古洛糖酸多糖或其可药用盐。
本发明的再一个目的是提供一种治疗肿瘤的方法。
根据本发明的一个方面， 提供了一种结构如下列通式（ I )所示的硫酸酯 化聚古洛糖酸多糖或其可药用盐：
在本发明的由通式（ I )表示的硫酸酯化聚古洛糖酸多糖或其可药用盐中， 所述古洛糖酸多糖由 L-古洛糖醛酸通过 1, 4糖苷键连接而成，其还原端 1位 为羟基， 糖环 C-2位完全硫酸酯化， C-3位部分硫酸酯化。
上述通式（I ) 中， n为 0或 1-23的整数， 例如 0、 1、 2、 3、 4、 5、 6、 7、 8、 9、 10、 11、 12、 13、 14、 15、 16、 17、 18、 19、 20、 21、 22或 23;
优选地， n为 2-13的整数， 更优选 n为 3、 4、 5、 6、 7、 8、 9或 10， 最优选 n为 4、 5、 6、 7或 8。 优选地， 所述含石克量为 7-15重量％， 更优选 9-13重量 %。 本发明中， 四糖至十二糖（特别是六糖至十糖）和 /或含^ 量为 7-15重量 % (特别是 9-13重量％ )时的生物效果较佳，很可能是它们较易被机体细胞识 别和接受。
本发明中， 所述^ 酸酯化聚古洛糖酸多糖的可药用盐， 例如可以是这些 化合物的钠盐、 钾盐、 钙盐、 镁盐等， 其中优选钠盐。 所述的可药用盐可用 常规方法制得。
根据本发明的另一方面， 提供了上述硫酸酯化聚古洛糖酸多糖或其可药 用盐的制备方法， 其包括：
将下列结构式（Π )所示的古洛糖醛酸多糖与横化试剂反应， 再经还原 剂还原， 形成通式（I )所示的硫酸酯化聚古洛糖酸多糖，
本发明中， 优选地， 所述横化剂可为氯横酸； 优选地， 横化反应温度可 为 45_85 °C， 更优选 60_75 °C， 反应时间可为 1. 5-4. 5 小时， 更优选 2-3. 5 小时， 最优选 3小时； 优选地， 所述还原剂可为硼氢化钠、 氰基硼氢化钠、
镍氢试剂或面素类还原剂等等。
根据本发明的又一个方面，提供了一种硫酸酯化聚古洛糖酸多糖或其可 药用盐在制备肿瘤生长和 /或转移抑制剂中的用途。
本发明中， 术语 "肿瘤" 可指包括恶性肿瘤在内的任何形式的肿瘤， 例 如， 所述的肿瘤可为肝癌、 胃癌、 结直肠癌、 肺癌、 乳腺癌、 胰腺癌、 肾癌、 膀胱癌、 前列腺癌、 黑色素瘤、 脑瘤等。
本发明中， 优选地， 所述硫酸酯化聚古洛糖酸多糖或其可药用盐可被用 作肿瘤生长抑制剂、 肿瘤转移抑制剂、血管生成抑制剂、 乙酰肝素酶抑制剂、
C-Me t酶抑制剂、 微管聚合抑制剂、 肌动蛋白解聚因子活性抑制剂、 和 /或肌 动蛋白聚集抑制剂。
根据本发明的还一个方面， 提供了一种药物组合物， 其包含治疗有效量 的本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐。 优选地， 所述药物组合 物中的活性成分由一种或多种本发明的硫酸酯化聚古洛糖酸多糖或其可药用 盐组成。 在另一实施方案中， 本发明的药物组合物中除了本发明的硫酸酯化 聚古洛糖酸多糖或其可药用盐外， 还可以含有一种或多种抗肿瘤药物或抗肿 瘤辅助药物作为或活性成分。
本发明中， 优选地， 所述药物组合物可以进一步包含可药用载体。 所述 的可药用载体可为本领域中通常使用的。
本发明中， 优选地， 所述药物组合物可以用作肿瘤生长抑制剂。
本发明中， 优选地， 所述药物组合物可以用作肿瘤转移抑制剂。
此外， 优选地， 所述药物组合物可以用作血管生成抑制剂、 乙酰肝素酶 抑制剂及 C-Me t酶抑制剂、微管聚合抑制剂、肌动蛋白解聚因子活性抑制剂、 和 /或肌动蛋白聚集抑制剂。
根据本发明的再一个方面， 提供了一种治疗肿瘤的方法， 其包括向需要 治疗的对象施用治疗有效量的本发明的硫酸酯化聚古洛糖酸多糖或其可药用
。
本发明中， 术语 "有效量" 可指为实现预期的效果所需的剂量和时段的 有效的量。 此有效量可能因某些因子而产生不同的变化， 如疾病的种类或治 疗时疾病的病症、 被施用的特定标的器官的构造、 病人个体大小、 或疾病或 症状的严重性。 本领域具有通常知识者不需要过度实验即可凭经验决定特定 化合物的有效量。
因此， 将本发明的硫酸酯化聚古洛糖酸多糖或其可药用盐用于制备肿瘤 治疗药物和肿瘤转移治疗药物，对于解决当前肿瘤治疗缺乏有效药物的困难， 具有特别重要的意义。
附图说明
图 1示出本发明的硫酸酯化聚古洛糖酸多糖各组分的柱分离图。
图 2示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌 MDA-MB-435原位 移植瘤的生长抑制作用。
图 3示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌 MDA-MB-435原位 移植瘤肺转移的抑制作用。 其中， A为 H&E染色显示肺上转移灶的典型照片
(放大倍数： 200 X ); B为 PGAS对 MDA-MB-435原位移植瘤肺转移抑制作用 的定量图。图中数据表示为一次典型实验的均值士 SD。 *p < 0. 05 , **p < 0. 01, 治疗组与对照组进行比较。
图 4示出本发明的硫酸酯化聚古洛糖酸多糖对人乳腺癌 MDA-MB-435原位 移植瘤血管生成的抑制作用。 其中， A为 CD31染色的典型照片， 箭头指向阳 性处（放大倍数： 200 X ) (图 A中： "C" 为对照组， "P" 为 PGAS处理组）； B 是 PGAS对人乳腺癌 MDA-MB-435原位移植瘤血管生成抑制作用的定量图。 图 中数据表示为一次实验的均值± 80。 **P < 0. 01， 治疗组与对照组进行比较。
图 5示出本发明的硫酸酯化聚古洛糖酸多糖对鼠黑色素瘤细胞 B16F1()实验
性肺转移的抑制作用。 其中， A为肺上转移灶的典型照片； B是 PGAS对 BwF^ 实验性肺转移抑制作用的定量图。图中数据表示为一次典型实验的均值士 SD。 相似的结果可以从至少两次独立实马全获得。 *P < 0. 05 , **p < 0. 01, 治疗组 与对照组进行比较。
图 6示出本发明的硫酸酯化聚古洛糖酸多糖抑制鸡胚尿嚢膜（ CAM )新生 血管生成。 其中， A是溶媒对照组； B是 200 g/egg的 PGAS组； C为 400 μ g/egg的 PGAS组； D是 800 g/egg的 PGAS (放大倍数： 40倍）。
图 7 示出本发明的石克酸酯化聚古洛糖酸多糖抑制乙酰肝素酶活性的图 谱。 其中， A 示出本发明的硫酸酯化聚古洛糖酸多糖抑制乙酰肝素酶活性的 HPLC图谱； B示出根据 A图结果计算的本发明的硫酸酯化聚古洛糖酸多糖对 乙酰肝素酶活性的抑制率。
图 8示出本发明的硫酸酯化聚古洛糖酸多糖对无细胞体系微管蛋白聚合 的抑制作用。 其中， A示出时间依赖关系， B示出剂量依赖关系。
图 9示出本发明的硫酸酯化聚古洛糖酸多糖对无细胞体系肌动蛋白解聚 的抑制作用。
图 10 示出本发明的硫酸酯化聚古洛糖酸多糖抑制肌动蛋白解聚因子对 肌动蛋白的解聚 /剪切活性。 其中， ##P<0. 01 Cof i l in 组与对照组比较； **ρ<0. 01 药物组与 Cof i l in组比较。
图 11 示出本发明的硫酸酯化聚古洛糖酸多糖各糖组分抑制肿瘤转移的 活性。
具体实施方式
在下文中， 将通过实施例更加详细地阐明本发明。 但是， 提供下列实施 例仅仅是出于说明目的， 本发明的范围不限于此。
主要药品与试剂
古洛糖醛酸多糖 ( olyguluronic ac id ), 购自中国海洋大学兰太药业有
限责任公司， 相对于右旋糖酐的重均分子量为 lOOOODa; 曱酰胺， 氯横酸等 均由国药集团化学试剂公司提供， 均为分析纯试剂； 右旋糖酐分子量标准品 购自 Fluka公司； 阿霉素注射液（ Adriamycin, ADM), 浙江海门制药厂生产， 浙卫药准字 （ 1996 )第 135501号， 含量： 10mg/支， 溶剂： 生理盐水， 配置 方法： 每次给药前用生理盐水稀释到所需浓度； TSK gel2000SWXL, TSK gel G3000SWXL色语柱， 日本 T0S0H公司； Bio- Gel- P6， Bio- Gel- P10， Bio-Rad 公司产品； Sephadex G- 10， Sepharose CL-4B, Pharmacia 公司产品； 微管 蛋白 （tubulin), 肌动蛋白 （actin), 均购自 SIGMA公司。
实验仪器
NEXUS-470智能型红外光语仪， NIC0LET公司产品； DPX-300型核磁共 振波语仪， Bruker公司产品； 凝胶渗透色谱（GPC)， 北京龙智达有限公司产 品； UV-2102紫外可见分光光度计， 美国尤尼科公司产品。
实验动物
棵小鼠， BALB/cA， 18-22g, 由中国科学院上海药物研究所提供； C57BL/6小鼠， 6-7周龄， 由中国科学院上海动物中心提供；
新鲜种蛋， 购自上海申宝养鸡场。
实施例 1 硫酸酯化聚古洛糖酸多糖（PGAS)的制备
保持在 5°C以下，向 10ml曱酰胺中逐滴加入 3ml氯横酸，反应 20分钟后， 加入 lg古洛糖醛酸多糖， 在 65-70°C反应 3小时， 向反应产物中加入 2倍体 积的 80%乙醇溶液， 反复搅拌， 得到粘稠物质。 再加入 80%乙醇溶液， 重复以 上步骤 2次。倾去溶液，加水得到粘稠物质， 用 l%Na2C03溶液调节 pH为 7.0， 用 2倍体积的 95%乙醇溶液醇沉，得到的沉淀物在 5{T60°C烘干，得到古洛糖 醛酸多糖的¾酸酯化 †生物，将该 †生物配成 4mg/ml的醋酸钠溶液（ H7.0 ), 加入硼氢 4匕钠， 使成 50mM， 30-40°0^,© 30min, ；水浴终止反应， 以 0.1M醋 酸调节 pH值， 释放未反应的硼氢化钠， 调节 pH值中性， 乙醇反复沉淀洗涤，
干燥， 即得硫酸酯化聚古洛糖酸多糖（ PGAS )粗品。 将 PGAS粗品配成 1 0%的 溶液， 用 95%的乙醇溶液进行沉淀， 将沉淀用无水乙醇洗涤， 干燥后， 配成 5%溶液， 用 3μπι膜过滤除杂质， 在 Sephadex G-10柱（ 15 χ 100cm )上进行 脱盐， 流动相为水， 分步收集， 洗脱液用¾酸-咔唑法检测， 合并含糖组分， 减压浓缩并除盐， 冷冻干燥， 即得精制的硫酸酯化聚古洛糖酸多糖。
釆用氧瓶燃烧法测定上述制备的硫酸酯化聚古洛糖酸多糖的含硫量。 取 约 25mg试样， 精密称定， 按照氧瓶燃烧法进行有机破坏， 选用 1000ml燃烧 瓶，以浓过氧化氢溶液 0. lml与水 1 0ml为吸收液，待生成的烟雾完全吸收后， 置冰浴中 15 分钟， 加热緩緩煮沸 2 分钟， 冷却， 加乙醇-醋酸铵緩冲液 (pH3. 7) 50ml , 乙醇 30ml， 0. 1%茜素红溶液 0. 3ml为指示液， 用高氯酸钡滴 定液（0. 05mo l /L)滴定至淡橙红色。 每 lml高氯酸钡滴定液（0. 05mo l /L)相当 于 1. 603mg 的 S。 测定结果发现， 按干燥品计算， 硫酸酯化聚古洛糖酸多糖 的含硫量为 11. 2重量％。
实施例 2 硫酸酯化聚古洛糖酸多糖（PGAS )的结构鉴定
对上述硫酸酯化聚古洛糖酸多糖的制备中得到的流分所含糖组分进行 结构鉴定。
1. 紫外吸收图谱
将上述硫酸酯化聚古洛糖酸多糖稀释至合适浓度， 以蒸馏水作为空白， 用 UV-2102紫外可见分光光度计在 190nm-400nm间扫描， 发现该流分在紫外 区无特异性吸收峰， 说明结构中无共轭双键结构。 但是在 190-200讓处有非 特异性吸收。
2. 红外光谱测定
取 0. 5mg的 PGAS， 用 KBr压片， 通过 NEXUS-470 智能型红外光语仪进 行红外光谱测定。 结果发现， 在 3219. 53cm—1处有羟基的对称伸缩振动， 1612. 58 cm— 1有羧酸盐的叛基对称伸缩振动， 1414. 33 cm— 1有羟基的剪式振动，
1103.97 cm— 1有羧基碳氧键的对称伸缩振动， 823.30 cm— 1有 C-0-S的伸缩振 动峰； 1274.62 cm—1有硫酸化后硫酸酯基中的 S=0伸缩振动峰， 表明该化合 物含有羧基、 羟基和 酸基的骨架结构。
3. PGAS核磁共振波谱测定
釆用 Bruker Auance DPX-300型核磁共振波语仪测定了 PGAS核磁共振碳 谱（13C-醒 R)。 结果发现， 谱图中基本看不到未横化的 C-2信号峰， 而仍然存 在未横化的 C-3信号峰。 表明 C-2位羟基硫酸酯化比较完全， C-3位羟基只 有部分被硫酸酯化。
4. PGAS的分子量与分子量分布
釆用 GPC方法对 PGAS的分子量进行测定。 使用的分子量标准品为 Fluka 公司的右旋糖酐， 色语柱为 TSK gel2000SWXL柱， 流动相为含 0.2%叠氮钠和 2.84%Na2S04的水溶液， 流速为 0.5ml/min， 测定温度在 35°C， 进样量为 25 μ 1, 使用 Refractive Index Detector 检测器进行检测。 结果发现， PGAS 相对于右旋糖酐的重均分子量为 2513Da，对多批 PGAS样品测定的结果表明， 其相对于右旋糖酐的重均分子量在 1500-8500Da。
综合以上检测结果， 确定上述流分为还原端 1位为羟基的聚古洛糖醛酸 的 C-2位完全硫酸酯化、 C-3位部分硫酸酯化得到的硫酸酯化聚古洛糖酸多 糖， 具有下列化学结构式（I ):
实施例 3 PGAS各组分的分离制备
取上述制备的 PGAS 样品， 配成 5%溶液， 用 3μπι膜过滤除杂质， 在
Bio- Gel- P6凝胶柱（ 1.6 χ 180cm)上进行分离， 流动相为 0.2mol/L NH4HC03, 分步收集， 洗脱液用 酸-咔唑法检测， 收集各含糖组分， 外水体积组分继续 上 Bio_Gel_P10凝胶柱（1.6x 180cm)分离，冷冻干燥，得系歹 'J PGAS糖组分， 经质语鉴定， 制备了 PGAS 的 2、 3、 4、 5、 6、 7、 8、 9、 10、 11及大于 11 糖的组分（图 1 )。 本发明下述各实验中，除实施例 12和 13使用上述实施例 3制备的产品 外， 其余实施例 4- 11均使用上述实施例 1制备的产品。
实施例 4 硫酸酯化聚古洛糖酸多糖（ PGAS )抑制肿瘤生长的疗效评价 将处于对数生长期的浓度为 2.5 X 107细胞 /毫升的人乳腺癌 MDA-MB-435 细胞（来源于美国典型培养物保藏中心（American Type Culture Collection, ATCC, Rockville, MD, USA)), 接种于 4-5周龄的雌性棵小鼠 (BALB/cA, 由 中国科学院上海药物研究所提供）左侧第二乳头脂肪垫。 当肿瘤体积生长到 100-200 mm3时， 将动物按瘤体积平均分为阴性对照组、 阿霉素注射液
( Adriamycin, ADM)给药组 (阳性对照组）和 PGAS 5 mg/kg及 20 mg/kg给药 组（PGAS实验组），每组 20只， PGAS实验组和阳性对照组每周静脉注射给药一 次， 连续给药 7周， 阴性对照组给以等量生理盐水。 实验釆用游标卡尺测量瘤 径， 每周二次， 肿瘤体积（V)按以下公式计算：
V = 1/2 X a b2
其中， a和 b分别表示肿瘤的长和宽。 根据测量的结果计算出相对肿瘤体 积（relative tumor volume, RTV ) ， 计算公式为： RTVt = Vt /V。。 其中， V。 为分笼给药时（即 d。）测量所得肿瘤体积， Vt为测量当天的肿瘤体积。 抗肿瘤 活性的药效学评价指标为相对肿瘤增殖率 T/C ( % ) ， 计算公式如下：
T/C (%) = 驅
CRTV
TRTV: 治疗组 RTV; CRTV: 阴性对照组 RTV。 疗效评价标准： T/C % >60 %为 无效； T/C % 60%， 并经统计学处理， P<0. 05为有效。
停止给药 1周后处死动物。 将肺组织固定于 Bouin' s 液（饱和苦味酸： 曱醛：冰醋酸 = 75: 25: 5 ) 24小时以上， 再用无水乙醇浸泡至肺组织转移灶呈 现为白色结节， 肺组织恢复为正常颜色。 在解剖显微镜下观测记录每个肺脏 的转移结节数。 部分原位瘤组织置于液氮中冻存， 以供抽提总 RNA和蛋白质。 部分肺脏、 原位瘤组织釆用 10%福尔马林固定液固定， 以 H&E染色和免疫组化 测定肿瘤组织中血管的生成情况。
结果发现，接种肿瘤后 15-20天，人乳腺癌 MDA-MB-435移植瘤瘤体积生 长至 100-200 mm3左右， 接种成活率在 100%; 接种后第 8周至第 9周荷瘤棵 小鼠肺组织出现大量转移灶， 转移率为 100%， 此时， 处死动物并进行抗肿瘤 药效学评价。 PGAS按每周一次静脉注射，连续 7周的治疗方案， PGAS 5 mg/kg 处理组即显示抑制原位瘤生长的能力， 但抑瘤效果不明显， T/C值为 67. 3%; PGAS 20mg/kg处理组则显示能够显著抑制人乳腺癌 MDA-MB-435棵小鼠原位 移植瘤的生长， T/C值达 37. 6%。 阳性对照药 ADM显示能够显著抑制人乳腺癌 MDA-MB-435原位瘤的生长， ADM 5mg/kg处理组的 T/C值为 21. 8%。 由此说明， PGAS能够显著抑制人乳腺癌 MDA-MB-435原位移植瘤的生长（图 2 )。 PGAS在 5 mg/kg , 20 mg/kg 每周静脉注射一次连续给药七周的剂量下， 对人乳腺癌 MDA-MB-435 原位移植瘤肺转移的抑制率分别为 60. 2%、 88. 4%。 阿霉素 ( 5mg/kg )对肺转移抑制率为 89. 8%。 说明 PGAS 能够显著抑制人乳腺癌 MDA-MB-435原位移植瘤的肺转移（图 3 )。进一步以免疫组织化学染色评价了 PGAS对体内血管生成的影响。 内皮细胞特异性标记物 CD31检测表明人乳腺 癌 MDA-MB-435原位移植瘤内有大量小血管生成。 PGAS 5 mg/kg处理组与对 照组相比移植瘤内小血管数无明显变化， PGAS 20 mg/kg 处理组与对照组相 比移植瘤内小血管数明显减少， 抑制率达 42. 1%。 说明 PGAS可显著抑制人乳
腺癌 MDA-MB-435原位移植瘤内血管生成（图 4 )。
实施例 5 硫酸酯化聚古洛糖酸多糖（ PGAS )抑制肿瘤转移的疗效评价 将 6-7周龄 C57BL/6小鼠 （由中国科学院上海动物中心提供） 随机分为生 理盐水阴性对照组、 PGAS 5 mg/kg及 20 mg/kg给药组， 每组各 10只动物。 将 处于对数生长期的浓度为 2. 5 X 106细胞 /毫升鼠黑色素瘤细胞 。（来源于美 国典型培养物保藏中心 （ Amer ican Type Cul ture Co l lect ion, ATCC, Rockvi l le, MD, USA ) )接种于小鼠尾静脉。 给细胞前 20分钟， PGAS组腹腔 注射给药一次， 阴性对照组给以等量生理盐水。 11天后取肺， 将肺组织固定 于 Bouin' s 液（饱和苦味酸：曱醛：冰醋酸 = 75: 25: 5 ) 24小时以上， 在解剖 显微镜下观测记录每个肺脏的转移结节数。 或再于无水乙醇中浸泡两天后， 待肺组织恢复为正常颜色再观察。 结果发现接种后 11天， 小鼠肺组织出现大 量转移灶， 转移率为 100%， 而按腹腔注射一次给药的方案， PGAS 5mg/kg处理 组即显示明显的抑制肿瘤转移的能力， 抑制率达 40. 81%， PGAS 20mg/kg处理 组则显示能极显著抑制鼠黑色素瘤细胞 。的肺转移， 抑制率达 82. 20%。 结 果显示 PGAS能显著抑制鼠黑色素瘤细胞 。的肺转移 （图 5 ) 。 本发明人还对 PGAS的作用机制进行了研究。
实施例 6 PGAS抑制血管生成试验
将新鲜种蛋（购自上海申宝养鸡场）置于 39 °C、 湿度为 50%的孵化器 Rol l-X base (Lyon Electr i c Company, CA， USA)中 （气室端向上）。 连续孵 育 7天后， 先用光照鉴定鸡胚是否存活， 并确定尿嚢膜所在位置， 作好标记， 在鸡蛋气嚢所在一端扎一个小孔，横放鸡胚（尿嚢膜朝上）， 用洗耳球轻吸气 嚢，使尿嚢膜塌陷与蛋壳分开，静置一段时间， 然后在鸡胚上方划一 1cm2见 方的开窗位置， 用剪刀磨切开窗， 吹去蛋壳上磨切的粉尘， 揭去开窗处的蛋 壳， 尿嚢膜清晰可见。 将最终作用浓度为 200、 400、 800 g/egg的 PGAS 10
μ 1 加在 0. 25 x 0. 25 x 0. 25 (长 x宽 x高） cm3明胶海绵上， 同时设置溶媒对 照， 然后将海绵轻轻地放在尿嚢膜无大血管的部位。 用灭菌透明胶布封好小 窗， 继续孵育 48小时， 揭开胶布进行观察， 拍照 （放大倍数： 40 X )， 评价 PGAS对鸡胚尿嚢膜血管生成的抑制作用。 结果发现（图 6 )， 在含有 PGAS海 绵旁边的尿嚢膜， 其血管密度明显下降， 并且呈浓度依赖性的抑制趋势。 表 明 PGAS具有抑制血管生成的作用。
实施例 7 PGAS抑制乙酰肝素酶（ heparanase )活性试验
利用从人胎盘中得到的人乙酰肝素酶的 cDNA， 通过 PCR扩增和基因重 组，构建了乙酰肝素酶的无血清昆虫表达系统和亲和柱纯化系统，获得了 95% 以上纯度的高活性乙酰肝素酶。 在 150 μ 1 反应緩冲液（50 mM 醋酸钠， pH4. 2 ) 中， 加入 0. 5 μ δ FITC-HS (异石克氰酸荧光素标记的乙酰肝素）和终 浓度为 25ng/ml的乙酰肝素酶， 同时加入不同浓度的 PGAS，在 37 °C反应 3 h。 100 °C 5 min后 10000 rpm离心 20 min以沉淀不溶物。 上清液用 0. 45 μ πι的 滤膜过滤后注射到 TSK ge l G3000SWXL柱上，上样量为 20 μ 1，緩冲液为 50mM Tr i s/ 150 mM NaCl , pH7. 5 , 流速为 0. 8ml/min。 用荧光检测器检测 FITC-HS 产物的荧光强度（Ex: 485讓， Em: 538nm )。 酶的相对活性通过完整 FITC-HS 前半峰面积的下降来评价，从而确定 PGAS对乙酰肝素酶活性的影响。结果发 现， PGAS呈剂量依赖性地抑制乙酰肝素酶活性， IC5。为 6. 55 ng/ml， 当剂量 为 40 ng/ml时， 其活性优于阳性对照物肝素 （hepar in ) (图 7 )。
实施例 8 PGAS抑制 C-Met酶活性试验
将酶反应底物 Poly (Glu, Tyr)包被酶标板， 加入 ATP溶液及一定浓度的 硫酸酯化聚古洛糖酸多糖和 c-Met酶液， 37°C摇床反应 1小时后，加入含 BSA 5mg/ml 的 T-PBS稀释 PY99抗体， 37°C摇床反应 0. 5小时， 再加入辣根过氧 化物酶标记羊抗鼠的 IgG， 37°C摇床继续反应 0. 5 小时， 最后加入 2mg/ml 的 0PD显色液， 25°C避光反应 1-10分钟后， 加入 2M H2S04中止反应， 酶标
仪测 490讓处的吸光度值， 计算酶活抑制率： 删 、 n 化合物 OD值 -无酶对照孔 OD值 、 ^
翻率 (%> = Q _阴性对照孔 OD值 -无酶对照孔 OD值 ) Χ 100 结果发现， 酸酯化聚古洛糖酸多糖在 lO g/ml浓度下对 c-Met的酶 活抑制率为 71.1%， 表明硫酸酯化聚古洛糖酸多糖具有抑制 c-Met酶活的作 用。
实施例 9 PGAS抑制微管蛋白 （ tubul in )聚合试验
96孔板中加入 10 μΐ不同浓度的 PGAS， 在 37 °C中预热 10 min。 微管 蛋白冻干粉用 PEM緩冲液 ( 100 mM PIPES, 1 mM MgCl2, 1 mM EGTA )， 另加 1 mM GTP， 5% 甘油稀释成浓度 12 μΜ， 加 90 μΐ于 96孔板中， 开启酶标仪， 设置温度为 37°C，检测波长为 340讓，每分钟混匀读取一次，连续测定 30 min。 结果表明， PGAS能明显抑制无细胞体系微管蛋白的聚合， 当 PGAS的浓度从 2.5 μΜ增加到 40 μΜ 时， 其抑制率从 18.26% 增加到 73.44%， 呈剂量依赖 性， 其 IC50 为 8.58 μΜ (图 8 )。
实施例 10 PGAS抑制肌动蛋白 （actin)解聚
先将 Pyrenyl标记的肌动蛋白加入聚集緩冲液 ( 100 mMTris-HCl, 20 mM MgCl2, 500 mM KC1, 2 mM CaCl2, pH 7.5) 37°C孵育 30 min, 使其聚集成纤 丝， 然后加入肌动蛋白解聚緩冲液（ 10 mM Tris_HCl， 0.2 mM CaCl2, 0.2 mM ATP, pH 8.0)及不同浓度的 PGAS， 将其稀释使其解聚， 设置荧光酶标仪， 使发射光波长为 360 nm， 吸收光波长为 410 nm， 温度 37°C， 每分钟混匀读 取一次， 连续测定 30min。 结果表明， PGAS能显著地抑制无细胞体系肌动蛋 白的解聚过程， 且呈剂量依赖性（ IC5。为 10.6 μΜ) (图 9 )。
实施例 11 PGAS抑制 cofilin解聚 /剪切肌动蛋白的活性
将硫酸酯化聚古洛糖酸多糖与 Sepharose CL-4B进行偶联， 制备了硫酸
酯化聚古洛糖酸多糖的亲和层析柱， 利用该层细柱， 对^ 酸酯化聚古洛糖酸 多糖在肺癌细胞株 A549上的结合蛋白进行了提取、分离，通过质谱鉴定发现 cofilin是与硫酸酯化聚古洛糖酸多糖结合较强的蛋白之一， 进一步的研究 发现硫酸酯化聚古洛糖酸多糖能明显抑制 cofilin对肌动蛋白的解聚 /剪切 活性（图 10)。说明硫酸酯化聚古洛糖酸多糖能与 cofilin结合，抑制 cofilin 的解聚 /剪切肌动蛋白的活性， 从而发挥抑制肿瘤细胞细胞迁移的作用。
实施例 12 PGAS各糖组分的活性测定
对实施例 3中制备的各 PGAS糖组分的抗肿瘤活性进行了测试， 结果发 现， PGAS 4-12糖（通式（ I ) 中 n=2-10)组分在静脉注射剂量小于 15mg/kg 时对人乳腺癌 MDA-MB-435棵小鼠原位移植瘤生长的 T/C值达 30%以下，对其 肺转移的抑制率达 95%以上， 6-10糖（通式（I ) 中 n=4-8)组分在剂量小于 10mg/kg时对人乳腺癌 MDA-MB-435棵小鼠原位移植瘤生长的 T/C值达 30%以 下，对其肺转移的抑制率达 95%以上，并以含石充量在 7 - 15重量％时效果更好， 9- 13重量％最好。
实施例 13 PGAS各糖组分抑制肿瘤转移活性测定
用 Transwell小室实险检测了实施例 3中分离得到的 PGAS不同糖组分 对肿瘤转移的影响。 胰酶消化体外传代对数期生长的乳腺癌 MDA-MB-435 细 胞， 用无血清培液洗涤细胞三次并稀释细胞至 2 X 107ml。 Transwell小室中 每孔上室加入 100 μ 1的细胞稀释液， 下室加入 600 μ 1含 10% FBS的培液， 上下室均加入 100 g/ml的 PGAS不同糖组分。 在含 5%C02的培养箱中 37。 C 孵育 12h后弃去培养液， 90%酒精溶液固定细胞 30min。 用 0.1%结晶紫溶液 (0.1M硼酸、 0.1% (w/v)结晶紫、 2%乙醇）于常温染色 10min， 清水漂净， 用 棉签拭去上层未迁移的细胞， 显微镜下观察和拍照并记录； 最后用 10%乙酸 溶液 100 μ 1/孔抽提 10m i η， 595nm测定 0D值， 计算各糖组分对乳腺癌细胞 的迁移抑制率。
迁移抑制率 （％ ) = ( 1-0D 处理组 /0D 对照组 ) χ 100%
结果发现， 对照组的 MDA-MB-435细胞在 12h之内可以沿 FBS的浓度梯 度自由的从上室迁移到下室， 从结晶紫染色后照相的结果可以看出， PGAS各 糖组分（从 4糖（通式 I中， n=2 )至 11糖（通式 I中， n=9 )及大于 11糖 组分）可以明显抑制肿瘤细胞迁移， 2糖组分（通式 I中， n=0 )、 3糖组分（通 式 I中， n=l )抑制率较低（图 11 )， 说明 PGAS各糖组分（特别是， 从 4糖 至 11糖及大于 11糖组分） 均具有抑制肿瘤细胞转移的作用。 统计学处理
以上数据应用 Sta tview统计处理软件进行统计分析， 结果以 "均值士标 准误" （Mean va lues士 SE )表示， 釆用方差分析 ( AN0VA )进行比较。
根据上述药理结果， 用常规的制剂手段将有效量的本发明的硫酸酯化聚 古洛糖酸多糖与药用载体混合， 可以制备药学组合物。 所述药学组合物可以 是肿瘤治疗药物和肿瘤转移治疗药物， 也可以是血管生成抑制剂、 乙酰肝素 酶抑制剂、 C-Met 酶抑制剂及微管聚合抑制剂和肌动蛋白解聚抑制剂。 将本 发明的硫酸酯化聚古洛糖酸多糖用于制备肿瘤治疗药物和肿瘤转移治疗药物 具有特别重要的意义。
Claims
1、 一种硫酸酯化聚古洛糖酸多糖或其可药用盐， 其中各个 L-古洛糖醛酸单 元通过 1, 4糖苷键连接， 其还原端 1位为羟基， 糖环 C-2位完全硫酸酯化。
2、 一种结构如下列通式（ I )所示的硫酸酯化聚古洛糖酸多糖或其可药用盐：
通式（ I ) 中， n表示 0或 1-23的整数， ^为 S03H, R2各自独立地为 H 或 S03H, 条件是： 硫酸酯化程度换算成硫酸酯化聚古洛糖酸多糖的含硫量， 为 5-20重量％。
3、、 根据权利要求 2所述的硫酸酯化聚古洛糖酸多糖或其可药用盐， 其中， 通式（ I ) 中， n为 2-13的整数， 优选为 3、 4、 5、 6、 7或 8。
4、、 根据权利要求 2或 3所述的硫酸酯化聚古洛糖酸多糖或其可药用盐， 其 中， 所述含^ ^量为 7-15重量％, 优选为 9-13重量％。
5、权利要求 2所述的硫酸酯化聚古洛糖酸多糖或其可药用盐的制备方法，其 包括：
将下列结构式（I I ) 所示的古洛糖醛酸多糖与磺化试剂反应， 再经还原 剂还原， 形成通式（I )所示的硫酸酯化聚古洛糖酸多糖，
6、 根据权利要求 5所述的制备方法， 其中， 所述磺化剂为氯磺酸； 磺化反应 温度为 45- 85 °C ,优选 60-75 °C ;反应时间为 1. 5-4. 5小时，优选 2-3. 5小时， 最优选 3小时； 和 /或， 所述还原剂为硼氢化钠、 氰基硼氢化钠、 镍氢试剂或 卤素类还原剂。
7、权利要求 1-4中任一项所述的硫酸酯化聚古洛糖酸多糖或其可药用盐在制 备肿瘤生长和 /或转移抑制剂中的用途。
8、 根据权利要求 7所述用途， 其中， 所述肿瘤选自肝癌、 胃癌、 结直肠癌、 肺癌、 乳腺癌、 胰腺癌、 肾癌、 膀胱癌、 前列腺癌、 黑色素瘤、 脑瘤。
9、权利要求 1-4中任一项所述的硫酸酯化聚古洛糖酸多糖或其可药用盐在制 备肿瘤生长抑制剂、 肿瘤转移抑制剂、血管生成抑制剂、 乙酰肝素酶抑制剂、 C-Me t酶抑制剂、 微管聚合抑制剂、 肌动蛋白解聚因子活性抑制剂、 和 /或肌 动蛋白聚集抑制剂中的用途。
10、 一种药物组合物， 其包含治疗有效量的权利要求 1-4任一项所述的5克酸 酯化聚古洛糖酸多糖或其可药用盐。
11、 根据权利要求 10所述的药物组合物在制备肿瘤生长和 /或转移抑制剂中 的用途。
12、根据权利要求 10所述的药物组合物在制备肿瘤生长抑制剂、肿瘤转移抑 制剂、 血管生成抑制剂、 乙酰肝素酶抑制剂、 C-Me t 酶抑制剂、 微管聚合抑 制剂、 肌动蛋白解聚因子活性抑制剂、 和 /或肌动蛋白聚集抑制剂中的用途。
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| ES14819337.8T ES2674976T3 (es) | 2013-07-02 | 2014-07-02 | Polisacárido de ácido poligulónico sulfatado o sal farmacéutica del mismo, método de preparación y uso del mismo |
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| DK14819337.8T DK3018147T3 (en) | 2013-07-02 | 2014-07-02 | SULPHATED POLYGULONIC ACID POLYSACCHARIDE OR PHARMACEUTICAL SALTS THEREOF, PROCEDURE FOR PREPARING THEREOF AND USING THEREOF |
| CA2916749A CA2916749C (en) | 2013-07-02 | 2014-07-02 | Sulfated polygulonic acid polysaccharide or pharmaceutical salt thereof, preparation method therefor and use thereof |
| JP2016522246A JP6757249B2 (ja) | 2013-07-02 | 2014-07-02 | 硫酸化ポリグルロン酸多糖又はその薬学的塩、その調製方法及びその使用 |
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| BR112015032849-0A BR112015032849B1 (pt) | 2013-07-02 | 2014-07-02 | Polissacarídeo de sulfato de ácido poligulônico ou sal de grau farmaceuticamente aceitável do mesmo, método de preparação do mesmo e composição farmaceutica |
| US14/901,297 US10058566B2 (en) | 2013-07-02 | 2014-07-02 | Sulfated polygulonic acid polysaccharide or pharmaceutical salt thereof, preparation method therefor and use thereof |
| CN201480038171.1A CN105358582B (zh) | 2013-07-02 | 2014-07-02 | 一种硫酸酯化聚古洛糖酸多糖或其可药用盐及其制备方法和用途 |
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| HK16109038.7A HK1220984A1 (zh) | 2013-07-02 | 2016-07-28 | 種硫酸酯化聚古洛糖酸多糖或其可藥用鹽及其製備方法和用途 |
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| CN107904240A (zh) * | 2017-12-04 | 2018-04-13 | 辽东学院 | 一种抗肝癌细胞转移的抑制剂组合物 |
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| EP3018147A1 (en) | 2016-05-11 |
| CN105358582A (zh) | 2016-02-24 |
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| CN105358582B (zh) | 2018-09-21 |
| PT3018147T (pt) | 2018-07-17 |
| US10058566B2 (en) | 2018-08-28 |
| MX2016000043A (es) | 2016-05-31 |
| EP3018147B1 (en) | 2018-05-23 |
| TR201809674T4 (tr) | 2018-07-23 |
| EA028655B1 (ru) | 2017-12-29 |
| US20160367591A1 (en) | 2016-12-22 |
| ES2674976T3 (es) | 2018-07-05 |
| EA201690123A1 (ru) | 2016-07-29 |
| ZA201509353B (en) | 2017-08-30 |
| JP6772318B2 (ja) | 2020-10-21 |
| CA2916749C (en) | 2021-03-09 |
| CA2916749A1 (en) | 2015-01-08 |
| DK3018147T3 (en) | 2018-07-23 |
| SG11201510717SA (en) | 2016-01-28 |
| JP2016523301A (ja) | 2016-08-08 |
| PL3018147T3 (pl) | 2018-10-31 |
| BR112015032849B1 (pt) | 2021-09-14 |
| JP2019090056A (ja) | 2019-06-13 |
| HUE038981T2 (hu) | 2018-12-28 |
| JP6757249B2 (ja) | 2020-09-16 |
| AU2014286711B2 (en) | 2018-05-10 |
| HK1220984A1 (zh) | 2017-05-19 |
| KR20160029817A (ko) | 2016-03-15 |
| KR102258569B1 (ko) | 2021-05-28 |
| EP3018147A4 (en) | 2017-04-05 |
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| AU2014286711A1 (en) | 2016-02-04 |
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