WO2019149179A1 - 糖胺聚糖衍生物及其制备方法和用途 - Google Patents

糖胺聚糖衍生物及其制备方法和用途 Download PDF

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WO2019149179A1
WO2019149179A1 PCT/CN2019/073572 CN2019073572W WO2019149179A1 WO 2019149179 A1 WO2019149179 A1 WO 2019149179A1 CN 2019073572 W CN2019073572 W CN 2019073572W WO 2019149179 A1 WO2019149179 A1 WO 2019149179A1
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occurrence
glycosaminoglycan derivative
independently selected
glycosaminoglycan
formula
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PCT/CN2019/073572
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English (en)
French (fr)
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任丽鸽
王景文
金学文
张利利
林森茂
李锂
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深圳市海普瑞药业集团股份有限公司
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Priority to CN201980011282.6A priority Critical patent/CN111670038B/zh
Priority to US16/966,951 priority patent/US11225531B2/en
Publication of WO2019149179A1 publication Critical patent/WO2019149179A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • 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
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • 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
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0078—Degradation products

Definitions

  • the present invention relates to glycosaminoglycan derivatives (in particular, glycosaminoglycan carboxylated derivatives), to processes for their preparation, and to their use for inhibiting tumor growth and/or metastasis.
  • glycosaminoglycan derivatives in particular, glycosaminoglycan carboxylated derivatives
  • Heparanase also known as heparanase, is a beta-glucuronidase that cleaves heparan sulfate (HS) in heparan sulfate proteoglycans, such as multi-ligand poly Sugar-1 (syndecan-1), thereby releasing growth factors that bind heparan sulfate.
  • HS heparan sulfate
  • proteoglycans such as multi-ligand poly Sugar-1 (syndecan-1)
  • Heparanase is highly expressed in most human tumor cells and significantly increases the angiogenic and migration capacity of tumor cells. Studies have demonstrated that elevated heparanase levels are associated with advanced progression and metastasis of many tumor types. For example, high levels of heparanase are associated with shorter postoperative survival times in patients. The direct role of heparanase in tumor metastasis has been demonstrated in the laboratories of Vlodavsky and Sanderson.
  • heparanase In addition to enzymatic functions, including the release of HS-bound growth factors and the degradation of extracellular matrix (ECM), heparanase also has a non-enzymatic function that affects tumor behavior and the microenvironment.
  • ECM extracellular matrix
  • Heparin is a linear polydisperse sulfated polysaccharide in the glycosaminoglycan family that has anticoagulant and antithrombotic activity.
  • the sugar chain of heparin consists of alternating uronic acid and D-glucosamine.
  • the main repeating unit is 2-O-sulfated L-iduronic acid (IdoA2S) ⁇ (1 ⁇ 4) and N-,6 -O-disulfated D-glucosamine (GlcN6S); less components are non-sulfated L-iduronic acid and D-glucuronic acid, and N-acetyl D-glucosamine and N-, 3-O-,6-O-trisulfated D-glucosamine (Casu B., 2005. "Structure and active domains of heparin.” In: Chemistry and biology of heparin and heparan sulfate. Amsterdam: Elsevier. 28; Casu B.
  • Heparin is effective in inhibiting heparanase, but in the heparanase inhibition strategy, it is impossible to use heparin at a high dose due to its anticoagulant activity.
  • LMWH low molecular weight heparin
  • the prior art has disclosed a class of non-antagonizing heparins which are useful as heparanase inhibitors, most of which comprise structurally engineered non-sulfated uronic acid residues which can be broken by glycosaminoglycans.
  • the 2-position-3 position carbon-carbon bond (diol cleavage) on the residue is obtained by ring opening of the glycoside ring.
  • Carboxylated derivatives of glycosaminoglycans and their use against tumors are disclosed in CN105744940A.
  • the present invention provides a glycosaminoglycan derivative which has both anti-tumor growth and anti-tumor metastasis activity, and particularly has good anti-tumor metastatic activity.
  • a first aspect of the invention provides a glycosaminoglycan derivative comprising a structural unit of formula (I), a structural unit of formula (IV) and a structural unit of formula (V):
  • R 2m , R 2n and R 2x are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • R 3n is each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • R 4m , R 4n and R 4x are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence;
  • the glycosaminoglycan derivative has a weight average molecular weight of 7000-14000 Da, preferably 8000-13500 Da, such as 8500-13000 Da, 8500-12500 Da or 9000-12500 Da;
  • the glycosaminoglycan derivative has a degree of uronic acid ring opening of 25% to 80%, preferably 25 to 60%.
  • a second aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a glycosaminoglycan derivative of the invention and one or more pharmaceutically acceptable carriers, preferably a solid preparation , semi-solid preparation, liquid preparation or gaseous preparation.
  • a third aspect of the invention provides the use of a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for inhibiting tumor growth and/or metastasis.
  • a fourth aspect of the invention provides a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention for use in inhibiting tumor growth and/or metastasis.
  • a fifth aspect of the invention provides a method of inhibiting tumor growth and/or metastasis comprising administering to an individual in need thereof an effective amount of a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention.
  • a sixth aspect of the invention provides a process for the preparation of a glycosaminoglycan derivative of the invention.
  • Figure 1 shows the survival rate of mice after administration of test samples.
  • degree of sulfation refers to a sulphur-to-carboxy ratio (SO 3 - /COO - molar ratio) as determined by conductometric titration according to Casu B. and Gennaro U., 1975, Carbohydr Res 39, 168-176. .
  • carboxyl increment refers to the ratio of the degree of sulfation of the starting material to the degree of sulfation of the carboxylated derivative. More specifically, the degree of sulfation of the raw material is the degree of sulfation determined by reducing the sample of the glycosaminoglycan intermediate obtained after the first oxidation step with NaBH4.
  • uronic acid ring opening degree refers to the number of ring-opened uronic acid residues per total uronic acid residue
  • uronic acid epoxide means formula (V) The ratio of the uronic acid structural unit having an epoxy structure to the total uronic acid residue.
  • Their detection and calculation references Guerrini, M., Guglieri, S., Naggi, A., Sasisekharan, R., & Torri, G. (2007).
  • Low molecular weight heparins Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy. The nuclear magnetic method in in Thrombosis and Hemostasis, 33, 478-487.
  • the invention provides a glycosaminoglycan derivative comprising a structural unit of formula (I), a structural unit of formula (IV), and a structural unit of formula (V):
  • R 2m , R 2n and R 2x are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • R 3n is each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • R 4m , R 4n and R 4x are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence;
  • the glycosaminoglycan derivative has a weight average molecular weight of 7000-14000 Da, preferably 8000-13500 Da, such as 8500-13000 Da, 8500-12500 Da or 9000-12500 Da;
  • the glycosaminoglycan derivative has a degree of uronic acid ring opening of 25% to 80%, preferably 25 to 60%.
  • the invention provides a glycosaminoglycan derivative further comprising a structural unit of formula (II):
  • R 2t and R 3t are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence.
  • the invention provides a glycosaminoglycan derivative further comprising a structural unit of formula (III):
  • R 2p is each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence.
  • the invention provides a glycosaminoglycan derivative further comprising a structural unit of formula (VI):
  • R 2r and R 3r are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence.
  • the invention provides a glycosaminoglycan derivative further comprising a structural unit of formula (VII):
  • R 2s , R 3s and R 4s are each independently selected from H and -SO 3 - (1/q E q+ ) at each occurrence;
  • E is each independently selected from H, an alkali metal (preferably lithium, sodium, potassium, rubidium or cesium), an alkaline earth metal (preferably magnesium or calcium) and aluminum;
  • an alkali metal preferably lithium, sodium, potassium, rubidium or cesium
  • an alkaline earth metal preferably magnesium or calcium
  • q are each independently an integer of 1, 2 or 3 at each occurrence.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise m units of the formula (I), wherein m is selected from any integer from 1 to 30, inclusive; Any integer selected from 1-20, inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise t structural units of the formula (II), wherein t is selected from any integer from 0 to 26, inclusive; Any integer selected from 0-8, inclusive; more preferably any integer selected from 1-8, inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise p units of the structural unit of formula (III), wherein p is selected from any integer from 0 to 26, inclusive; Any integer selected from 0-4, inclusive; more preferably any integer selected from 1-4, inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise n structural units of formula (IV), wherein n is selected from any integer from 1 to 24, inclusive; Any integer selected from 1-18, inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise x units of the formula (V), wherein x is selected from any integer from 0 to 18, inclusive; Any integer selected from 0-8, inclusive; more preferably any integer selected from 1-8, inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise r structural units of the formula (VI), wherein r is selected from any integer from 0-26 (eg, 1-26) , including end values; preferably any integer selected from 0-4 (eg, 1-4), inclusive.
  • the one or more polysaccharide chains of the glycosaminoglycan derivative may each comprise s of the structural units of the formula (VII), wherein s is selected from any integer from 1 to 26, inclusive; Any integer selected from 1-8, inclusive.
  • each structural unit is arranged in any order.
  • E q+ is H + , Li + , Na + , K + , Mg 2+ , Ca 2+ or Al 3+ .
  • the number of structural units of formula (I) in the glycosaminoglycan derivative is greater than the sum of the number of structural units of formula (II) and formula (III).
  • the sum of the number of structural units of formula (I) and the number of structural units of formula (I), formula (II) and formula (III) in the glycosaminoglycan derivative The ratio is greater than 0.9.
  • the ratio of the number of structural units of the formula (I) to the sum of the number of structural units of the formula (I) and the formula (IV) in the glycosaminoglycan derivative is 0.3- 0.7.
  • the sum of the number of structural units of formula (V) and the number of structural units of formula (I), formula (IV) and formula (V) in the glycosaminoglycan derivative The ratio is less than 0.1.
  • the uronic acid epoxidation is less than 25%.
  • the molecular weight distribution of the glycosaminoglycan derivative is as follows:
  • the molecular weight distribution of the glycosaminoglycan derivative is as follows:
  • the glycosaminoglycan derivative has a sulfonium carboxyl ratio of from 0.80 to 1.65.
  • the glycosaminoglycan derivative has a sulfonium carboxyl ratio of from 1.0 to 1.4.
  • the invention encompasses any combination of the various embodiments.
  • the invention provides a method of making the glycosaminoglycan derivative, comprising the steps of:
  • step b) optionally, hydrolyzing the epoxidized product obtained in step a), preferably under neutral conditions;
  • step d) further oxidizing the product from step c) under conditions effective to convert the dialdehyde to a carboxyl group, preferably without nitrous oxide, preferably by chlorite (preferably sodium chlorite) )get on.
  • chlorite preferably sodium chlorite
  • the method further comprises a 2N-desulfation step of a glucosamine residue, which is carried out before step a), after step a) or after step b), said desulfation step comprising
  • the pyridine is salted and then stirred in a mixed solvent of DMSO and water or methanol.
  • the glycosaminoglycan is native heparin or synthetic heparin (optionally chemically or enzymatically modified) from any animal and organ source, preferably selected from optionally 2-O- and / or 2-N-desulfated heparin, unfractionated heparin, low molecular weight heparin (LMWH, molecular weight 3,500-8,000 Da) and heparan sulfate (HS) having a sulfonate of 0.8-2.8, preferably 0.9-2.5 More preferably, it is selected from unfractionated heparin and LMWH which are optionally 2-O- and/or 2-N-desulfated.
  • LMWH low molecular weight heparin
  • HS heparan sulfate
  • the glycosaminoglycan has a weight average molecular weight of from 10,000 Da to 30,000 Da, preferably from 15,000 Da to 25000 Da, such as from 15,000 Da to 20,000 Da, from 15,000 Da to 19,000 Da or from 17,000 Da to 19,000 Da.
  • the heparin chain can naturally comprise from about 5% to 35% of a 2-O-non-sulfated uronic acid residue, from 0% to 50% of an N-acetylated glucosamine residue, and from about 0% to about -6. % of N-unsubstituted (neither N-sulfated or N-acetylated) glucosamine residues.
  • the degree of different degrees of sulfation depends on the source of heparin (animal species, source of the organ) and extraction operations.
  • Each 2-O- or 2N-non-sulfated residue of glycosaminoglycan (without a 3-O-sulfate substituent) is susceptible to oxidation under ring opening (cleavage) as well as ortho diols and OH/NH 2 conversion to aldehydes.
  • fractional 2-O-desulfation of the starting glycosaminoglycan allows adjustment of the ratio of glucosamine/uronic acid cleavage residues.
  • the glycosaminoglycan derivative exhibits a carboxyl number increase of from 1.3 to 2.0, wherein the carboxyl number is increased by the ratio of the degree of sulfation of the starting material to the degree of sulfation of the glycosaminoglycan derivative.
  • the degree of sulfation of the raw material is the degree of sulfation measured after the sample of the glycosaminoglycan intermediate obtained after the first oxidation step (step c)) is reduced with NaBH 4 .
  • Specific procedures for calculating the carboxyl number increase can be found in CN105744940A.
  • compositions and methods of treatment are provided.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a glycosaminoglycan derivative of the invention and one or more pharmaceutically acceptable carriers, preferably a pharmaceutical composition A solid preparation, a semisolid preparation, a liquid preparation or a gaseous preparation.
  • the pharmaceutical composition may further comprise one or more additional therapeutic agents.
  • the invention provides the use of a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for inhibiting tumor growth and/or metastasis.
  • the invention provides a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention for use in inhibiting tumor growth and/or metastasis.
  • the invention provides a method of inhibiting tumor growth and/or metastasis comprising administering to an individual in need thereof an effective amount of a glycosaminoglycan derivative of the invention or a pharmaceutical composition of the invention.
  • the tumor is a solid tumor, a hematological tumor, or a soft tissue tumor; preferably a solid tumor, such as breast cancer, pancreatic cancer, bladder cancer, prostate cancer, colon cancer, gastric cancer, or lung cancer.
  • “Pharmaceutically acceptable carrier” in the context of the present invention means a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting humans and/or within the scope of sound medical judgment. Tissues of other animals without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, minerals. Oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol and the like.
  • the composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents as needed.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).
  • compositions of the invention may act systemically and/or locally.
  • they may be administered in a suitable route, for example by injection (for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or by oral, buccal, or oral administration.
  • injection for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation
  • transdermal administration or by oral, buccal, or oral administration.
  • compositions of the invention may be administered in a suitable dosage form.
  • the dosage forms include, but are not limited to, tablets, capsules, troches, hard candy, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions. Injectable solutions, elixirs, syrups.
  • an effective amount refers to an amount of a derivative that, after administration, will relieve to some extent one or more symptoms of the condition being treated.
  • the dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent need for treatment. It is noted that the dose value can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. It is to be further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or the composition of the supervised composition.
  • glycosaminoglycan derivative of the invention administered will depend on the individual being treated, the severity of the condition or condition, the rate of administration, the handling of the glycosaminoglycan derivative, and the judgment of the prescribing physician.
  • an effective dose will be from about 0.0001 mg to about 50 mg per kg body weight per day, for example from about 0.01 mg/kg/day to about 10 mg/kg/day (single or divided doses). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, such as from about 0.7 mg/day to about 700 mg/day.
  • a dose level that is not higher than the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any harmful side effects, provided that the larger The dose is divided into several smaller doses to be administered throughout the day.
  • the glycosaminoglycan derivative of the present invention may be included in the pharmaceutical composition in an amount or in an amount of from about 0.01 mg to about 1000 mg, suitably from 0.1 to 500 mg, preferably from 0.5 to 300 mg, more preferably from 1 to 150 mg, particularly preferably from 1 to 50 mg. For example, 1.5 mg, 2 mg, 4 mg, 10 mg, 25 mg, and the like.
  • treating means reversing, alleviating, inhibiting the progression of a condition or condition to which such a term applies or the progression of one or more symptoms of such a condition or condition, or preventing such A condition or condition or one or more symptoms of such condition or condition.
  • “Individual” as used herein includes human or non-human animals.
  • Exemplary human individuals include a human individual (referred to as a patient) or a normal individual having a disease, such as the disease described herein.
  • “Non-human animals” in the present invention include all vertebrates, such as non-mammals (eg, birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (eg, sheep, dogs). , cats, cows, pigs, etc.).
  • compositions of the invention may also comprise one or more additional therapeutic or prophylactic agents.
  • step 2) Hydrolysis ring opening: the temperature of the solution in step 1) is raised to 70 ° C, and the mixture is hydrolyzed under neutral conditions for 24 h. After the reaction is completed, the temperature is lowered to room temperature, and the solution sample is taken to detect the molecular weight; the dialysis is desalted, and the intermediate product is dried by rotary evaporation. The product weight was 50 g, the yield was 100%, and Mw was 16034 Da.
  • step 2) Hydrolysis ring opening: Weigh 6.4g of step 1) freeze-dried product, add 96mL purified water, raise the solution to 70 ° C, hydrolyze under neutral conditions for 24h, then settle to room temperature after concentration, concentrate and freeze to obtain ring Oxystructure ring opening product.
  • the increment is 1.75, and the degree of opening of the uronic acid is 41.2%.
  • a 1:1 HCl solution prepared from a commercially available 36%-38% concentrated hydrochloric acid to purified water in a volume ratio of 1:1).
  • a 1:1 HCl solution prepared from a commercially available 36%-38% concentrated hydrochloric acid to purified water in a volume ratio of 1:1
  • Comparative Example (Since the weight average molecular weight of the starting materials is not illustrated in CN105744940A, those skilled in the art are unable to obtain their products for comparison.
  • This comparative example is obtained by repeating the operations of Example 1, Example 4 and Example 8 in CN105744940A.
  • the product has a slightly different characterization parameter from the product of Example 8 in CN105744940A)
  • N/A means not detected.
  • products H1301 and H1302 are significantly different from the products obtained by the process in CN105744940A (especially in terms of weight average molecular weight and uronic acid ring opening).
  • the prepared products were tested from the viewpoints of in vitro heparanase inhibition, anti-cancer growth, anti-cancer metastasis, and acute toxicity, respectively.
  • the in vitro heparanase inhibitory activity of the product prepared in the present application was examined by referring to the method for detecting heparanase (heparanase-like) inhibitory activity in vitro in CN105744940A. The results are shown in the table below.
  • the MM.1S cells were cultured in vitro in an RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and cultured at 37 ° C 5% CO 2 . Passaged three times a week. When the cells are in the exponential growth phase, the cells are harvested, counted, and 100 ⁇ L of cell suspension containing 3 ⁇ 104 tumor cells (cell suspension in RPMI 1640 medium plus 10% heat-inactivated fetal bovine serum, cell viability greater than 95%) ) inoculated into 96-well plates.
  • a sample prepared from RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum was added to each of the 6 wells to a final concentration of 100 ⁇ g/mL, and an equal volume was added to the other 6 wells.
  • RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum for measuring cell absorbance at 0 ⁇ g/mL
  • six medium wells except for cells and samples completely Culture medium
  • add 20 ⁇ L of CCK8 reagent to all wells incubate for 1-2 hours at 37 ° C under 5% CO 2 , and measure the absorbance at 450 nm under a microplate reader.
  • the effect of the sample on cell growth was calculated by taking the absorbance value of the well of the complete medium as the background and the cell absorbance at 0 ⁇ g/mL as 100%.
  • Inhibition rate (cell absorbance at 0 ⁇ g/mL - absorbance of sample well) / (cell absorbance at 0 ⁇ g/mL - absorbance at complete medium) (the above values are the average of the corresponding 6 wells)
  • Hela cells were cultured in vitro, cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, and cultured at 37 ° C 5% CO 2 . Passaged three times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and 100 ⁇ L of cell suspension containing 2*10 4 tumor cells (cell suspension in DMEM medium without fetal bovine serum, cell viability greater than 95%) was inoculated into Transwell. In the chamber, 600 ⁇ L of DMEM medium containing 10% heat-inactivated fetal bovine serum was added to the small chamber. The test samples were dissolved in the medium inside and outside the chamber to a final concentration of 100 ⁇ g/mL.
  • Migration inhibition rate (average of control cell migration cells - average of migrated cells under sample treatment) / average value of control cell migration cells
  • 4T1 cells were cultured in vitro in an RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and cultured at 37 ° C 5% CO 2 . Passaged three times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and 50 ⁇ L of cell suspension containing 1*10 5 tumor cells (cell suspension in RPMI 1640 medium without fetal bovine serum) was subcutaneously inoculated into the fourth fat pad of the abdomen. The mice used were BALB/c mice, female, 6-8 weeks old, weighing 18-22 grams. Randomly grouped according to body weight and tumor inoculation sequence, 12 mice per group. The test sample group was administered the next day after the inoculation.
  • the negative control group was given an equal amount of physiological saline.
  • the tumor in situ was surgically removed 12 days after the tumor was inoculated, and was not administered on the same day, and continued to be administered as described above the next day after surgery.
  • Murine melanoma B16 cells were cultured in vitro in an RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and cultured at 37 ° C 5% CO 2 . Passaged three times a week. When the cells were in the exponential growth phase, the cells were harvested, counted, and formulated into a suspension containing 2.5*10 6 cells/mL with PBS. SPF-grade healthy C57/BL6 mice weighing 18-20 g were grouped into 8 groups of mice and divided into negative control group, model group and experimental group.
  • test samples were injected into the tail vein of the experimental group at a dose of 2.5 mg/kg mouse body (dissolved in physiological saline at a concentration of 0.25 mg/mL), and the tail vein of the negative control group and the model group was as small as 10 ⁇ L/g. Rats were injected with normal saline. The injection time was recorded in turn. After 30 minutes, 200 ⁇ L of cell suspension containing 5*10 5 tumor cells (cell suspension in PBS, cell viability greater than 90%) was injected into the tail vein of the model group and the experimental group, negative. The control group was injected with the same amount of normal saline in the tail vein. Animals were monitored daily and body weights were recorded. All mice were sacrificed at the same time points on days 12-14 after cell injection, lung tissues were taken and fixed in Bouin solution, and tumor metastasis sections on the lung surface were counted under a stereo microscope.
  • Inhibition rate of B16 metastasis of test samples (median of lung metastasis of model group - median lung metastasis of experimental group) / median of lung metastasis of model group *100%

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Abstract

公开了一种糖胺聚糖羧基化衍生物、其制备方法，及其用于抑制肿瘤生长和/或转移的用途。

Description

糖胺聚糖衍生物及其制备方法和用途 技术领域
本发明涉及糖胺聚糖衍生物(特别地，糖胺聚糖羧基化衍生物)、其制备方法，及其用于抑制肿瘤生长和/或转移的用途。
背景技术
乙酰肝素酶，又名类肝素酶，是一种β-葡萄糖醛酸内切酶，其能够断裂硫酸乙酰肝素蛋白聚糖中的硫酸乙酰肝素(heparan sulfate，HS)，例如多配体聚糖-1(syndecan-1)，从而释放与硫酸乙酰肝素结合的生长因子。
乙酰肝素酶在大部分人肿瘤细胞中高表达，并显著增加肿瘤细胞的血管生成和迁移能力。研究已经证实升高的乙酰肝素酶水平与许多肿瘤类型的晚期进展和转移相关。例如，高水平的乙酰肝素酶与患者较短的手术后存活时间相关。在Vlodavsky和Sanderson的实验室已证明乙酰肝素酶在肿瘤转移中的直接作用。
除了酶促功能(包括释放与HS结合的生长因子和降解细胞外基质(ECM))，乙酰肝素酶还具有非酶促功能，其可影响肿瘤行为及微环境。
肝素是糖胺聚糖家族中的一种线形多分散的硫酸化多糖，其具有抗凝血和抗血栓形成活性。肝素的糖链由交替的糖醛酸和D-葡糖胺组成，主要的重复单元是2-O-硫酸化L-艾杜糖醛酸(IdoA2S)α(1→4)和N-，6-O-二硫酸化D-葡糖胺(GlcN6S)；较少组分是非硫酸化L-艾杜糖醛酸和D-葡萄糖醛酸，以及N-乙酰基D-葡糖胺和N-，3-O-，6-O-三硫酸化D-葡糖胺(Casu B.，2005.“Structure and active domains of heparin.”In：Chemistry and biology of heparin and heparan sulfate.Amsterdam：Elsevier.1-28；Casu B.和Lindahl U.，2001，“Structure and biological interactions of heparin and heparan sulfate.”Adv Carbohydr Chem Biochem，57：159-206)。肝素可有效地抑制乙酰肝素酶，但是在乙酰肝素酶抑制策略中，由于其抗凝血活性，将肝素以高剂量使用是不可能的。
有趣的是，比肝素更可被生物利用且较低抗凝血活性的低分子量肝素 (LMWH)似乎可能通过对肿瘤生长和转移的直接作用延长癌症患者的存活期。这可能至少部分由于对乙酰肝素酶活性的抑制(Zacharski L.R.和Lee，A.Y.2008.Heparin as an anticancer therapeutic.Expert Opin Investig Drugs 17：1029-1037)。
现有技术已经公开了一类可作为乙酰肝素酶抑制剂的非抗凝血肝素，其中大部分包含结构改造后的非硫酸化糖醛酸残基，此类结构可通过断裂糖胺聚糖残基上的2位-3位碳-碳键(二醇裂解)而使糖苷环开环获得。
CN105744940A中公开了糖胺聚糖的羧基化衍生物及其抗肿瘤的用途。
发明内容
本发明提供一种糖胺聚糖衍生物，其同时具有抗肿瘤生长和抗肿瘤转移活性，特别是具有良好的抗肿瘤转移活性。
本发明的第一方面提供糖胺聚糖衍生物，其包含式(I)的结构单元、式(IV)的结构单元和式(V)的结构单元：
Figure PCTCN2019073572-appb-000001
其中：
R 1m、R 1n和R 1x在每次出现时各自独立地选自H、-SO 3 -·(1/q E q+)和-(C＝O)CH 3，并且R 1m、R 1n和R 1x在每次出现时优选为-SO 3 -·(1/q E q+)或-(C＝O)CH 3；
R 2m、R 2n和R 2x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
R 3n在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
R 4m、R 4n和R 4x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；
q在每次出现时各自独立地为1、2或3的整数；
所述糖胺聚糖衍生物的重均分子量为7000-14000Da，优选为8000-13500Da，例如8500-13000Da、8500-12500Da或9000-12500Da；并且
所述糖胺聚糖衍生物的糖醛酸开环度为25％-80％，优选25-60％。
本发明的第二方面提供药物组合物，其包含预防或治疗有效量的本发明的糖胺聚糖衍生物以及一种或多种药学上可接受的载体，所述药物组合物优选为固体制剂、半固体制剂、液体制剂或气态制剂。
本发明的第三方面提供本发明的糖胺聚糖衍生物或者本发明的药物组合物在制备用于抑制肿瘤生长和/或转移的药物中的用途。
本发明的第四方面提供本发明的糖胺聚糖衍生物或者本发明的药物组合物，其用于抑制肿瘤生长和/或转移。
本发明的第五方面提供抑制肿瘤生长和/或转移的方法，所述方法包括向需要其的个体给药有效量的本发明的糖胺聚糖衍生物或者本发明的药物组合物。
本发明的第六方面提供本发明的糖胺聚糖衍生物的制备方法。
附图说明
图1显示给予测试样品后小鼠的生存率。
具体实施方式
本文中的术语“硫酸化程度”是指磺羧比(SO 3 -/COO -摩尔比率)，其通过根据Casu B.和Gennaro U.，1975，Carbohydr Res 39，168-176的电导滴定来测定。
本文中的术语“羧基增量”是指原料的硫酸化程度与羧基化衍生物的硫酸化程度的比率。更具体地，原料的硫酸化程度是将原料经第一个氧化步骤后获得的糖胺聚糖中间体的样品用NaBH4还原后测定的硫酸化程度。
本文中的术语“糖醛酸开环度”是指开环的糖醛酸残基数量/总的糖醛酸残基数量，并且术语“糖醛酸环氧度”是指式(V)的具有环氧结构的糖醛酸结构单元占总糖醛酸残基的比例。它们的检测和计算参考文献Guerrini，M.，Guglieri，S.，Naggi，A.，Sasisekharan，R.，&Torri，G.(2007).Low molecular weight heparins：Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy.Seminars in Thrombosis and Hemostasis，33，478-487中的核磁方法进行。
糖胺聚糖衍生物
在一些实施方案中，本发明提供糖胺聚糖衍生物，其包含式(I)的结构单元、式(IV)的结构单元和式(V)的结构单元：
Figure PCTCN2019073572-appb-000002
其中：
R 1m、R 1n和R 1x在每次出现时各自独立地选自H、-SO 3 -·(1/q E q+)和-(C＝O)CH 3，并且R 1m、R 1n和R 1x在每次出现时优选为-SO 3 -·(1/q E q+)或-(C＝O)CH 3；
R 2m、R 2n和R 2x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
R 3n在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
R 4m、R 4n和R 4x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；
q在每次出现时各自独立地为1、2或3的整数；
所述糖胺聚糖衍生物的重均分子量为7000-14000Da，优选为8000-13500Da，例如8500-13000Da、8500-12500Da或9000-12500Da；并且
所述糖胺聚糖衍生物的糖醛酸开环度为25％-80％，优选25-60％。
在优选的实施方案中，本发明提供糖胺聚糖衍生物，其还包含式(II)的结构单元：
Figure PCTCN2019073572-appb-000003
其中：
R 2t和R 3t在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
q在每次出现时各自独立地为1、2或3的整数。
在优选的实施方案中，本发明提供糖胺聚糖衍生物，其还包含式(III) 的结构单元：
Figure PCTCN2019073572-appb-000004
其中：
R 2p在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
q在每次出现时各自独立地为1、2或3的整数。
在优选的实施方案中，本发明提供糖胺聚糖衍生物，其还包含式(VI)的结构单元：
Figure PCTCN2019073572-appb-000005
其中：
R 2r和R 3r在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
q在每次出现时各自独立地为1、2或3的整数。
在优选的实施方案中，本发明提供糖胺聚糖衍生物，其还包含式(VII)的结构单元：
Figure PCTCN2019073572-appb-000006
其中：
R 1s在每次出现时各自独立地选自H、-SO 3 -·(1/q E q+)和-(C＝O)CH 3，并且优选为-SO 3 -·(1/q E q+)或-(C＝O)CH 3；
R 2s、R 3s和R 4s在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
q在每次出现时各自独立地为1、2或3的整数。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含m个所述式(I)的结构单元，其中m选自1-30的任意整数，包括端值；优选选自1-20的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含t个所述式(II)的结构单元，其中t选自0-26的任意整数，包括端值；优选选自0-8的任意整数，包括端值；更优选选自1-8的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含p个所述式(III)的结构单元，其中p选自0-26的任意整数，包括端值；优选选自0-4的任意整数，包括端值；更优选选自1-4的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含n个所述式(IV)的结构单元，其中n选自1-24的任意整数，包括端值；优选选自1-18的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含x个所述式(V)的结构单元，其中x选自0-18的任意整数，包括端值；优选选自0-8的任意整数，包括端值；更优选选自1-8的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含r个所述式(VI)的结构单元，其中r选自0-26(例如1-26)的任意整数，包括端值；优选选自0-4(例如1-4)的任意整数，包括端值。
任选地，所述糖胺聚糖衍生物的一条或多条多糖链可以各自包含s个所述式(VII)的结构单元，其中s选自1-26的任意整数，包括端值；优选选自1-8的任意整数，包括端值。
在本发明的糖胺聚糖衍生物的每条多糖链中，各结构单元以任意顺序排列。
在优选的实施方案中，E q+为H +、Li +、Na +、K +、Mg 2+、Ca 2+或Al 3+。
在优选的实施方案中，所述糖胺聚糖衍生物中的式(I)的结构单元的个数大于式(II)和式(III)的结构单元的个数的总和。
在优选的实施方案中，所述糖胺聚糖衍生物中的式(I)的结构单元的个数与式(I)、式(II)和式(III)的结构单元的个数的总和的比值大于0.9。
在优选的实施方案中，所述糖胺聚糖衍生物中的式(I)的结构单元的个数与式(I)和式(IV)的结构单元的个数的总和的比值为0.3-0.7。
在优选的实施方案中，所述糖胺聚糖衍生物中的式(V)的结构单元的个数与式(I)、式(IV)和式(V)的结构单元的个数的总和的比值小于0.1。
在优选的实施方案中，糖醛酸环氧度小于25％。
在优选的实施方案中，所述糖胺聚糖衍生物的分子量分布如下：
分子量范围(Da) 比例(重量％)
大于10000 15-80，优选25-80
6000-10000 15-50
小于6000 5-50
。优选地，所述糖胺聚糖衍生物的分子量分布如下：
分子量范围(Da) 比例(重量％)
大于10000 30-75
6000-10000 20-40
小于6000 5-30
或
分子量范围(Da) 比例(重量％)
大于10000 30-75
6000-10000 20-40
小于6000 0-30
。
在优选的实施方案中，所述糖胺聚糖衍生物的磺羧比为0.80-1.65。优选地，所述糖胺聚糖衍生物的磺羧比为1.0-1.4。
本发明涵盖各个实施方案的任意组合。
在一些实施方案中，本发明提供制备所述糖胺聚糖衍生物的方法，其包括以下步骤：
a)任选地，将糖胺聚糖的糖醛酸残基的C2、C3环氧化，其优选在碱性水溶液(优选氢氧化钠水溶液)中进行；
b)任选地，将步骤a)所得环氧化产物水解开环，其优选在中性条件下进行；
c)在将相邻的二醇并任选地将相邻的OH/NH 2有效转化为二醛的条件下，将糖胺聚糖的10％-100％(优选25％-100％)的2-O-并任选地将2N-，3-O-非硫酸化残基氧化，所述氧化优选通过高碘酸盐(优选高碘酸钠)进行；以及
d)在将所述二醛有效转化为羧基的条件下，且在无氮气保护下，将得自步骤c)的产物进一步氧化，所述进一步氧化优选通过亚氯酸盐(优选亚氯酸钠)进行。
在优选的实施方案中，所述方法还包括葡糖胺残基的2N-脱硫酸化步骤，该步骤在步骤a)之前，步骤a)之后或者步骤b)之后进行，所述脱硫酸化步骤包括与吡啶成盐，然后在DMSO与水或甲醇的混合溶剂中搅拌。
在优选的实施方案中，所述糖胺聚糖为来自任意动物和器官来源的天然肝素或者合成肝素(其任选地被化学或酶促修饰)，优选选自任选地2-O-和/或2-N-脱硫酸化的肝素、未分级肝素、低分子量肝素(LMWH，其分子量为3,500-8,000Da)和硫酸类肝素(HS)，其具有0.8-2.8，优选0.9-2.5的磺羧比；更优选选自任选地2-O-和/或2-N-脱硫酸化的未分级肝素和LMWH。
在优选的实施方案中，所述糖胺聚糖的重均分子量为10000Da至30000Da，优选为15000Da至25000Da，例如15000Da至20000Da、15000Da至19000Da或17000Da至19000Da。
作为一个实例，肝素链可天然地包含约5％至35％的2-O-非硫酸化糖醛酸残基、0％至50％的N-乙酰化葡糖胺残基和约0％-6％的N-未取代的(既未N-硫酸化也未N-乙酰化)葡糖胺残基。不同的硫酸化程度取决于肝素来源(动物种类、器官来源)以及提取操作。
糖胺聚糖的每个2-O-或2N-非硫酸化残基(不具有3-O-硫酸根取代基)易于进行开环(裂解)下的氧化以及邻位的二醇和OH/NH 2向醛的转化。任选地，起始糖胺聚糖的分级2-O-脱硫酸化允许调节葡糖胺/糖醛酸裂解残基的比率。
在优选的实施方案中，所述糖胺聚糖衍生物表现出1.3-2.0的羧基增量，其中所述羧基增量以原料的硫酸化程度与糖胺聚糖衍生物的硫酸化程度的比率的形式计算。更具体地，所述原料的硫酸化程度是将原料经第一个氧化步骤(步骤c))后获得的糖胺聚糖中间体的样品用NaBH 4还原后测定的硫酸化程度。用于计算羧基增量的具体操作可参见CN105744940A。
药物组合物和治疗方法
在一些实施方案中，本发明提供药物组合物，其包含预防或治疗有效量的本发明的糖胺聚糖衍生物以及一种或多种药学上可接受的载体，所述药物组合物优选为固体制剂、半固体制剂、液体制剂或气态制剂。在一些实施方案中，所述药物组合物还可包含一种或多种其它治疗剂。
在一些实施方案中，本发明提供本发明的糖胺聚糖衍生物或者本发明的药物组合物在制备用于抑制肿瘤生长和/或转移的药物中的用途。
在一些实施方案中，本发明提供本发明的糖胺聚糖衍生物或者本发明的药物组合物，其用于抑制肿瘤生长和/或转移。
在一些实施方案中，本发明提供抑制肿瘤生长和/或转移的方法，所述方法包括向需要其的个体给药有效量的本发明的糖胺聚糖衍生物或者本发明的药物组合物。
在一些实施方案中，所述肿瘤为实体瘤、血液肿瘤或软组织肿瘤；优选为实体瘤，例如乳腺癌、胰腺癌、膀胱癌、前列腺癌、结肠癌、胃癌或肺癌。
本发明中“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。
在本发明的药物组合物中可使用的药学上可接受的载体包括但不限于无菌液体，例如水和油，包括那些石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物通过静脉内给药时，水是示例性载体。还可以使用生理盐水和葡萄糖及甘油水溶液作为液体载体，特别是用于注射液。适合的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽糖、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等。所述组合物还可以视需要包含少量的湿润剂、乳化剂或pH缓冲剂。口服制剂可以包含标准载体，如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(1990)中所述。
本发明的药物组合物可以系统地作用和/或局部地作用。为此目的，它们可以适合的途径给药，例如通过注射(如静脉内、动脉内、皮下、腹膜内、 肌内注射，包括滴注)或经皮给药；或通过口服、含服、经鼻、透粘膜、局部、以眼用制剂的形式或通过吸入给药。
对于这些给药途径，可以适合的剂型给药本发明的药物组合物。
所述剂型包括但不限于片剂、胶囊剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、酏剂、糖浆剂。
如本文中所使用的术语“有效量”指被给药后会在一定程度上缓解所治疗病症的一或多种症状的衍生物的量。
可调整给药方案以提供最佳所需响应。例如，可给药单次推注，可随时间给药数个分剂量，或可如治疗情况的急需所表明而按比例减少或增加剂量。要注意，剂量值可随要减轻的病况的类型及严重性而变化，且可包括单次或多次剂量。要进一步理解，对于任何特定个体，具体的给药方案应根据个体需要及给药组合物或监督组合物的给药的人员的专业判断来随时间调整。
所给药的本发明的糖胺聚糖衍生物的量会取决于所治疗的个体、病症或病况的严重性、给药的速率、糖胺聚糖衍生物的处置及处方医师的判断。一般而言，有效剂量在每日每kg体重约0.0001mg至约50mg，例如约0.01mg/kg/日至约10mg/kg/日(单次或分次给药)。对70kg的人而言，这会合计为约0.007mg/日至约3500mg/日，例如约0.7mg/日至约700mg/日。在一些情况下，不高于前述范围的下限的剂量水平可以是足够的，而在其它情况下，仍可在不引起任何有害副作用的情况下采用较大剂量，条件是首先将所述较大剂量分成数个较小剂量以在一整天中给药。
本发明的糖胺聚糖衍生物在药物组合物中的含量或用量可以是约0.01mg至约1000mg，适合地是0.1-500mg，优选0.5-300mg，更优选1-150mg，特别优选1-50mg，例如1.5mg、2mg、4mg、10mg、25mg等。
除非另外说明，否则如本文中所使用，术语“治疗”意指逆转、减轻、抑制这样的术语所应用的病症或病况或者这样的病症或病况的一或多种症状的进展，或预防这样的病症或病况或者这样的病症或病况的一或多种症状。
如本文所使用的“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物) 和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
在一些实施方案中，本发明的药物组合物还可以包含一种或多种另外的治疗剂或预防剂。
实施例
以下列举实施例和实验例，进而详细地说明本发明，但它们不限制本发明的范围，另外在不脱离本发明的范围下可进行变化。
实施例1
1)C2、C3环氧化：称取未分级肝素5g(Mw＝18306Da)，溶于62.5g的1mol/L NaOH溶液中，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解36h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入100g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇10.0mL，继续搅拌1h，终止反应，透析24h(除盐)，得到透析液。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入100g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，用30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H0242。收率88.45％，Mw＝7001Da，SO 3 -/COO -＝0.85，羧基增量1.88，糖醛酸开环度73.1％。
实施例2
1)高碘酸钠氧化：称取未分级肝素5g(Mw＝17000Da)于250g水中，搅拌溶解。低温条件下加入100g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇10.0mL，继续搅拌1h，终止反应。透析24h，得到透析液。
2)亚氯酸钠氧化：取第1)步得到的透析液在低温条件下加入100g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h后，用30％(m/v)NaOH溶液调至中性，反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H0232。收率79.23％，Mw＝12810Da，SO 3 -/COO -＝1.55， 羧基增量1.22，糖醛酸开环度为26.3％。
实施例3
1)C2、C3环氧化：称取50g未分级肝素(Mw＝17000Da)，加入625.0g的1mol/L NaOH溶液，反应液升温至60℃附近，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解24h，反应结束后降至室温，取溶液样品检测分子量；透析除盐，旋蒸冻干得中间产品，干品重50g，收率100％，Mw＝16034Da。
3)高碘酸钠氧化：取第2)步得到的中间产品5g，溶于100g水中。在低温条件下，加入233g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌8h。反应结束后，加入乙二醇20.0mL，继续搅拌1h，终止反应，透析24h(除盐)，得到透析液。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入100g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，用30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H1011。收率89.90％，Mw＝9161Da，SO 3 -/COO -＝1.17，羧基增量1.68，糖醛酸开环度为43.1％。
实施例4
1)与实施例3步骤1)相同。
2)与实施例3步骤2)相同。
3)高碘酸钠氧化：取第2)步得到的中间产品5g，溶于100g水中。在低温条件下，加入116g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇20.0mL，继续搅拌1h，终止反应，透析24h(除盐)，得到透析液。
4)同实施例3中第4)步，得到的样品H1015，收率89.69％，Mw＝8717Da，SO 3 -/COO -＝1.08，羧基增量1.81，糖醛酸开环度为45.5％。
实施例5
1)C2、C3环氧化：称取未分级肝素8g(Mw＝18150Da)，加入1mol/L NaOH溶液(约102.4mL)，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积 比配制)调至中性，向反应液加入酒精进行醇沉，将沉淀溶解并冻干。
2)水解开环：称取第1)步冻干产品6.4g，加入96mL纯化水，溶液升温至70℃，中性条件下水解24h，反应结束后降至室温，浓缩、冻干，得到环氧结构开环产物。
3)高碘酸钠氧化：向第2)步环氧结构开环产物6.14g加入177mL纯化水溶解，在低温条件下加入等体积的0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇17.7mL，继续搅拌1h，终止反应。向反应液加入酒精进行醇沉(除盐)，将醇沉后所得上清液离心，合并沉淀，用少量水溶解，冻干，获得邻二醛中间体。
4)亚氯酸钠氧化：向邻二醛中间体(6g)加入579mL纯化水，反应液降温，加入6.03％(m/v)NaClO 2溶液120mL，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，用1mol/L NaOH溶液调至中性，分批加入亚硫酸氢钠固体中和亚氯酸钠。向反应液加入酒精进行醇沉，沉淀溶解后用截留分子量3500Da的再生纤维素透析袋透析，透析液旋蒸浓缩后冻干获得样品H7103，Mw＝11790Da，SO 3 -/COO -＝1.26，羧基增量1.75，糖醛酸开环度41.2％。
实施例6
1)C2、C3环氧化：称取未分级肝素(Mw＝17675Da)10g，溶于125g的1mol/L NaOH溶液中，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解24h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入200g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇20.0mL，继续搅拌1h，终止反应，透析24h(除盐)，得到透析液。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入200g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H8261，Mw＝10222Da，SO 3 -/COO -＝1.05，糖醛酸开环度57.6％。
实施例7
1)C2、C3环氧化：称取未分级肝素10g(Mw＝17806Da)，溶于125g 的1mol/L NaOH溶液中，反应液升温至60℃，反应60min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解24h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入200g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇20.0mL，继续搅拌1h，终止反应，将反应液透析24h。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入200g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H9053，Mw＝7112Da，SO 3 -/COO -＝1.13，糖醛酸开环度46.1％。
实施例8
1)C2、C3环氧化：称取未分级肝素10g(Mw＝18306Da)，溶于125g的1mol/L NaOH溶液中，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性，溶液透析16h。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解4h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入200g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇20.0mL，继续搅拌1h，终止反应，向反应液加入酒精进行醇沉(除盐)，将醇沉后所得上清液离心，合并沉淀，用少量水溶解。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入200g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H8073，Mw＝11781Da，SO 3 -/COO -＝1.38，羧基增量为1.41，糖醛酸开环度28.5％。
实施例9
1)C2、C3环氧化：称取未分级肝素5g(Mw＝17000Da)，溶于62.5g的1mol/L NaOH溶液中，反应液升温至60℃，反应30min；反应结束后，在室 温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解24h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入100g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇10.0mL，继续搅拌1h，终止反应，将反应液透析。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入100g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H9252，Mw＝8587Da，SO 3 -/COO -＝1.10，羧基增量为1.76，糖醛酸开环度46.6％。
实施例10
1)C2、C3环氧化：称取未分级肝素5g(Mw＝18306Da)，溶于62.5g的1mol/L NaOH溶液中，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性。
2)水解开环：将第1)步溶液升温至70℃，中性条件下水解24h，反应结束后降至室温。
3)高碘酸钠氧化：在低温条件下向第2)步反应液加入100g 0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇10.0mL，继续搅拌1h，终止反应，将反应液透析。
4)亚氯酸钠氧化：在低温条件下向第3)步得到的反应液加入100g的6.03％(m/v)NaClO 2溶液，用冰乙酸调pH至4.0左右，室温继续搅拌反应24h，30％(m/v)NaOH溶液调至中性。反应液透析24h，将透析液旋蒸浓缩后冻干获得样品H0123，Mw＝11445Da，SO 3 -/COO -＝1.21，羧基增量为1.64，糖醛酸开环度33.2％。
实施例11
1)C2、C3环氧化：称取未分级肝素4g(Mw＝18839Da)，加入1mol/L NaOH溶液(约51.2mL)，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比 配制)调至中性，将反应液透析72h，浓缩、冻干。
2)水解开环：称取第1)步冻干产品3g，加入45mL纯化水，溶液升温至70℃，中性条件下水解48h，反应结束后降至室温，浓缩、冻干，得到环氧结构开环产物。
3)高碘酸钠氧化：向第2)步环氧结构开环产物(2g)加入57.7mL纯化水溶解，在低温条件下加入等体积的0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇5.77mL，继续搅拌1h，终止反应。反应液在4℃下透析16h，浓缩，冻干，获得邻二醛中间体。
4)亚氯酸钠氧化：向邻二醛中间体(1g)加入100mL纯化水，反应液降温至0℃，加入6.03％(m/v)NaClO 2溶液20.6mL，用冰乙酸调pH至4.0，室温继续搅拌反应24h，用0.5mol/L NaOH溶液调至中性。反应液用截留分子量3500Da的再生纤维素透析袋透析16h，透析液旋蒸浓缩后冻干获得样品H1301，Mw＝7064Da，SO 3 -/COO -＝1.06，羧基增量1.75，糖醛酸开环度28.9％。
对比例(由于CN105744940A中未说明起始原料的重均分子量，本领域技术人员无法获得其产品以用于对比。该对比例为重复CN105744940A中实施例1、实施例4和实施例8的操作所得产品，其表征参数与CN105744940A中实施例8的产品略有差异)
1)C2、C3环氧化：称取未分级肝素4g(Mw＝18839Da)，加入1mol/L NaOH溶液(约51.2mL)，反应液升温至60℃，反应30min；反应结束后，在室温下冷却，用1∶1 HCl溶液(将市售36％-38％浓盐酸与纯化水按1∶1的体积比配制)调至中性，将反应液透析72h，浓缩、冻干。
2)水解开环：称取第1)步冻干产品3.0g，加入45mL纯化水，溶液升温至70℃，中性条件下水解48h，反应结束后降至室温，浓缩、冻干，得到环氧结构开环产物。
3)高碘酸钠氧化：向第2)步环氧结构开环产物(2g)加入57.7mL纯化水溶解，在低温条件下加入等体积的0.2mol/L NaIO 4溶液，避光，控温4℃，搅拌16h。反应结束后，加入乙二醇5.77mL，继续搅拌1h，终止反应。反应液在4℃下透析16h，浓缩，冻干，获得邻二醛中间体。
4)亚氯酸钠氧化：向邻二醛中间体(1g)加入100mL纯化水，反应液降温至0℃并在N 2气氛中搅拌条件下加入6.03％(m/v)NaClO 2溶液20.6mL，用 冰乙酸调pH至4.0，室温继续搅拌反应24h，在室温下通过流动的N 2搅拌另外3小时后，用0.5mol/L NaOH溶液调至中性。反应液用截留分子量3500 Da的再生纤维素透析袋透析16h，透析液旋蒸浓缩后冻干获得样品H1302，Mw＝6499Da，SO 3 -/COO -＝1.24，羧基增量1.34，糖醛酸开环度22.6％。
上述实施例与对比例所得产品的理化性质数据汇总如表1所示。
表1
样品编号 重均分子量(Da) 糖醛酸开环度 糖醛酸环氧度 磺羧比 羧基增量
H0242 7001 73.1％ 3.1％ 0.85 1.88
H0232 12810 26.3％ 0.3％ 1.55 1.22
H1011 9161 43.1％ 4.8％ 1.17 1.68
H1015 8717 45.5％ 4.8％ 1.08 1.81
H7103 11790 41.2％ 19.8％ 1.26 1.75
H8261 10222 57.6％ 3.3％ 1.05 N/A
H9053 7112 46.1％ 5.1％ 1.13 N/A
H8073 11781 28.5％ 21.3％ 1.38 1.41
H9252 8587 46.6％ 4.6％ 1.10 1.76
H0123 11445 33.2％ 17.6％ 1.21 1.64
H1301 7064 28.9％ 3.9％ 1.06 1.75
H1302 6499 22.6％ 4.7％ 1.24 1.34
N/A表示未检测。
所制备获得的产品的分子量分布数据如表2所示。
表2
样品编号 重均分子量 ＞10000 8000-10000 6000-8000 ＜6000
H0242 7001 17.6 13.9 20.3 48.2
H0232 12810 59.5 14.0 12.7 13.8
H1011 9161 36.2 17.9 19.6 26.3
H1015 8717 32.2 17.9 20.6 29.3
H7103 11790 56.0 17.0 14.2 12.8
H8261 10222 44.9 17.8 17.6 19.7
H9053 7112 18.3 15.5 22.0 44.2
H8073 11781 58.8 15.8 12.5 12.9
H9252 8587 31.4 17.7 20.8 30.1
H0123 11445 55.4 16.6 14.1 13.8
H1301 7064 18.0 14.6 21.1 46.3
H1302 6499 14.1 12.5 18.6 54.8
通过比较产品H1301和H1302可见，本申请的产品与通过CN105744940A中的工艺获得的产品显著不同(特别是在重均分子量和糖醛酸开环度上有显著差异)。
生物学实验
以下分别从体外乙酰肝素酶抑制、抗癌细胞生长、抗癌细胞转移以及急性毒性的角度对所制备的产品进行检测。
实验例1：体外乙酰肝素酶抑制检测
参照CN105744940A中体外检测乙酰肝素酶(类肝素酶)抑制活性的方法，检测本申请中所制备产品的体外乙酰肝素酶抑制活性。结果如下表中所示。
样品编号 乙酰肝素酶抑制活性(IC 50，ng/mL)
H0242 17.3
H0232 16.9
H1011 24.3
H7103 15.0
H8261 20.7
H8073 15.0
H9252 15.2
H0123 14.5
H1301 13.7
H1302 11.6
实验例2：抗MM.1S细胞生长实验
MM.1S细胞体外悬浮培养，培养条件为RPMI 1640培养基中加10％热灭活胎牛血清，37℃5％CO 2培养。一周三次传代处理。当细胞处于指数生长期时，收取细胞，计数，将含3×104个肿瘤细胞的100μL细胞悬液(细胞悬于RPMI 1640培养基中加10％热灭活胎牛血清，细胞活力大于95％)接种到96孔板中。细胞培养过夜后，向6个孔中分别加入用含10％热灭活胎牛血清的RPMI 1640培养基配制的样品，使样品终浓度为100μg/mL，向另外6个孔中分别加入等体积的含10％热灭活胎牛血清的RPMI 1640培养基(用于测定0μg/mL下的细胞吸光度值)，此外还设置六个除不加细胞和样品外其余均相同的培养基孔(完全培养基孔)，37℃5％CO 2培养48小时后向所有孔中加入20μL CCK8试剂，37℃5％CO 2下孵育1-2个小时，在酶标仪下检测450nm下的吸光度值，以完全培养基孔的吸光度值为背景，0μg/mL下的细胞吸光度值为100％，计算样品对细胞生长的影响。
抑制率＝(0μg/mL下的细胞吸光度值-样品孔吸光值)/(0μg/mL下的细胞吸光度值-完全培养基孔吸光值)(以上各值为相应6个孔的平均值)
实验结果如下表中所示。
Figure PCTCN2019073572-appb-000007
Figure PCTCN2019073572-appb-000008
结果显示：本发明的糖胺聚糖的羧基化衍生物能够显著地抑制MM.1S细胞的生长，且抑制活性与对比产品H1302相近。
实验例3：细胞迁移实验：Transwell实验(Hela细胞)
实验方法参考文献Dai X Y，Yan J，Fu X等人，Aspirin inhibits cancer metastasis and angiogenesis via targeting heparanase.[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research，2017，23(20)：6267进行。
Hela细胞体外贴壁培养，培养条件为DMEM培养基中加10％热灭活胎牛血清，37℃5％CO 2培养。一周三次传代处理。当细胞处于指数生长期时，收取细胞，计数，将含2*10 4个肿瘤细胞的100μL细胞悬液(细胞悬于无胎牛血清的DMEM培养基中，细胞活力大于95％)接种到Transwell小室中，在小室外加入600μL含10％热灭活胎牛血清的DMEM培养基。小室内外的培养基中均溶有测试样品，使其终浓度为100μg/mL，每个样品设置3个重复小室，另设3个不加药物的小室为对照，37℃5％CO 2培养24小时后，用预冷的95％乙醇固定30min，然后用棉签轻轻擦拭小室内表面并用PBS洗去未附着的细胞，用1％结晶紫溶液染色，晾干后拍照对比迁移的细胞数量。
迁移抑制率＝(对照孔迁移细胞的平均值-样品处理下迁移细胞的平均值)/对照孔迁移细胞的平均值
实验结果如下表所示。
Figure PCTCN2019073572-appb-000009
结果显示：本申请的糖胺聚糖的羧基化衍生物能够显著地抑制Hela细胞的迁移，在会发生恶性转移的肿瘤治疗中极具潜力。而对照样品H1302抑制Hela细胞迁移的活性较弱。
实验例4：原位4T1乳腺癌肺转移动物实验
4T1细胞体外悬浮培养，培养条件为RPMI 1640培养基中加10％热灭活胎牛血清，37℃5％CO 2培养。一周三次传代处理。当细胞处于指数生长期时，收取细胞，计数，将含1*10 5个肿瘤细胞的50μL细胞悬液(细胞悬于无胎牛血清的RPMI 1640培养液中)皮下接种于腹部第四脂肪垫，所用小鼠为BALB/c小鼠，雌性，6-8周龄，体重18-22克。根据体重和肿瘤接种顺序随机分组，每组12只小鼠。测试样品组接种第二天开始给药。每天两次腹腔给药，共20mg/kg剂量。阴性对照组给予等量生理盐水。接种肿瘤12天时手术切除原位肿瘤，当天不给药，术后第二天继续按照上述方式给药。
小鼠生存期：每只小鼠达到实验终点(体重降低大于20％)实施安乐死并记录生存期，绘制Kaplan-Meier生存曲线。
实验结果如下表及附图1中所示。
组别 阴性对照组 H1301 H1302 H7103 H0123
中位生存期(天) 38 46 40.5 51 64
相对对照组延长中位生存期(％)   21.1％ 6.6％ 34.2％ 68％
结果显示，本申请的衍生物H7103、H1301和H0123均能够显著地延长模型小鼠的中位生存期，尤其是H7103和H0123能够将模型小鼠的中位生存期分别延长34.2％和68％，对比样品H1302能够微弱地延长模型小鼠的中位生存期，但是与阴性对照组相比差异并不显著。
实验例5：B16鼠黑色素瘤肺转移实验
鼠黑色素瘤B16细胞体外贴壁培养，培养条件为RPMI 1640培养基中加10％热灭活胎牛血清，37℃5％CO 2培养。一周三次传代处理。当细胞处于指数生长期时，收取细胞，计数，用PBS配制成含2.5*10 6个细胞/mL的悬液。取体重18-20g的SPF级健康C57/BL6小鼠，按每组8只小鼠进行分组，分为阴性对照组、模型组和实验组。分别向实验组小鼠尾静脉按2.5mg/kg小鼠体重注射测试样品(溶解在生理盐水中，浓度为0.25mg/mL)，向阴性对照组和模型组小鼠尾静脉按10μL/g小鼠体重注射生理盐水。依次记录下注射时间，30分钟后，将含5*10 5个肿瘤细胞的200μL细胞悬液(细胞悬于PBS，细胞活力大于90％)注射到模型组和实验组小鼠尾静脉中，阴性对照组尾静脉注射等量生理盐水。每日监控动物并记录体重，细胞注射后第12-14天的同一时间点处死所有小鼠，取肺组织并固定在Bouin溶液中，在体视显微镜下对肺表面的肿瘤转移节计数。
测试样品对B16转移的抑制率＝(模型组肺转移节中位数-实验组肺转移节中位数)/模型组肺转移节中位数*100％
实验结果如下表中所示。
样品编号 H9053 H0242 H9252 H8073 H8261 H0123 H7103
抑制率(％) 25 18 37 90 85 93 92
结果显示，本申请的衍生物均有显著的抗肿瘤转移能力。
除本文中描述的那些外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的，这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (15)

  1. 糖胺聚糖衍生物，其包含式(I)的结构单元、式(IV)的结构单元和式(V)的结构单元：
    Figure PCTCN2019073572-appb-100001
    其中：
    R 1m、R 1n和R 1x在每次出现时各自独立地选自H、-SO 3 -·(1/q E q+)和-(C＝O)CH 3，并且R 1m、R 1n和R 1x在每次出现时优选为-SO 3 -·(1/q E q+)或-(C＝O)CH 3；
    R 2m、R 2n和R 2x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    R 3n在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    R 4m、R 4n和R 4x在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；
    q在每次出现时各自独立地为1、2或3的整数；
    所述糖胺聚糖衍生物的重均分子量为7000-14000Da，优选为8000-13500Da，例如8500-13000Da、8500-12500Da或9000-12500Da；并且
    所述糖胺聚糖衍生物的糖醛酸开环度为25％-80％，优选25-60％。
  2. 权利要求1的糖胺聚糖衍生物，其还包含式(II)的结构单元：
    Figure PCTCN2019073572-appb-100002
    其中：
    R 2t和R 3t在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
    q在每次出现时各自独立地为1、2或3的整数。
  3. 权利要求1或2的糖胺聚糖衍生物，其还包含式(III)的结构单元：
    Figure PCTCN2019073572-appb-100003
    其中：
    R 2p在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
    q在每次出现时各自独立地为1、2或3的整数。
  4. 权利要求1-3中任一项的糖胺聚糖衍生物，其还包含式(VI)的结构单元：
    Figure PCTCN2019073572-appb-100004
    其中：
    R 2r和R 3r在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
    q在每次出现时各自独立地为1、2或3的整数。
  5. 权利要求1-4中任一项的糖胺聚糖衍生物，其还包含式(VII)的结构单元：
    Figure PCTCN2019073572-appb-100005
    其中：
    R 1s在每次出现时各自独立地选自H、-SO 3 -·(1/q E q+)和-(C＝O)CH 3，并且优选为-SO 3 -·(1/q E q+)或-(C＝O)CH 3；
    R 2s、R 3s和R 4s在每次出现时各自独立地选自H和-SO 3 -·(1/q E q+)；
    E在每次出现时各自独立地选自H、碱金属(优选锂、钠、钾、铷或铯)、碱土金属(优选镁或钙)和铝；并且
    q在每次出现时各自独立地为1、2或3的整数。
  6. 权利要求1-5中任一项的糖胺聚糖衍生物，其中糖醛酸环氧度小于25％。
  7. 权利要求1-6中任一项的糖胺聚糖衍生物，其中所述糖胺聚糖衍生物的分子量分布如下：
    分子量范围(Da) 比例(重量％) 大于10000 15-80，优选25-80 6000-10000 15-50 小于6000 5-50
    优选地，所述糖胺聚糖衍生物的分子量分布如下：
    分子量范围(Da) 比例(重量％) 大于10000 30-75 6000-10000 20-40 小于6000 5-30
    或
    分子量范围(Da) 比例(重量％) 大于10000 30-75 6000-10000 20-40 小于6000 0-30
    。
  8. 权利要求1-7中任一项的糖胺聚糖衍生物，其中所述糖胺聚糖衍生物的磺羧比为0.80-1.65，优选1.0-1.4。
  9. 药物组合物，其包含预防或治疗有效量的权利要求1-8中任一项的糖胺聚糖衍生物以及药学上可接受的载体，所述药物组合物优选为固体制剂、半固体制剂、液体制剂或气态制剂。
  10. 权利要求1-8中任一项的糖胺聚糖衍生物在制备用于抑制肿瘤生长和/或转移的药物中的用途。
  11. 权利要求10的用途，其中所述肿瘤为实体瘤、血液肿瘤或软组织肿瘤；并且优选为实体瘤，例如乳腺癌、胰腺癌、膀胱癌、前列腺癌、结肠癌、胃癌或肺癌。
  12. 制备权利要求1-8中任一项的糖胺聚糖衍生物的方法，其包括以下步骤：
    a)任选地，将糖胺聚糖的糖醛酸残基的C2、C3环氧化，其优选在碱性水溶液(优选氢氧化钠水溶液)中进行；
    b)任选地，将步骤a)所得环氧化产物水解开环，其优选在中性条件下进行；
    c)在将相邻的二醇并任选地将相邻的OH/NH 2有效转化为二醛的条件下，将糖胺聚糖的10％-100％(优选25％-100％)的2-O-并任选地将2N-，3-O-非硫酸化残基氧化，所述氧化优选通过高碘酸盐(优选高碘酸钠)进行；以及
    d)在将所述二醛有效转化为羧基的条件下，且在无氮气保护下，将得自步骤c)的产物进一步氧化，所述进一步氧化优选通过亚氯酸盐(优选亚氯酸钠)进行；
    优选地，所述方法还包括葡糖胺残基的2N-脱硫酸化步骤，该步骤在步骤a)之前，步骤a)之后或者步骤b)之后进行，所述脱硫酸化步骤包括与吡啶成盐，然后在DMSO与水或甲醇的混合溶剂中搅拌。
  13. 权利要求12的方法，其中所述糖胺聚糖为来自任意动物和器官来源的天然肝素或者合成肝素(其任选地被化学或酶促修饰)，优选选自任选地2-O-和/或2-N-脱硫酸化的肝素、未分级肝素、低分子量肝素(LMWH)和硫 酸类肝素，其具有0.8-2.8，优选0.9-2.5的磺羧比；更优选选自任选地2-O-和/或2-N-脱硫酸化的未分级肝素和LMWH。
  14. 权利要求12或13的方法，其中所述糖胺聚糖的重均分子量为10000Da至30000Da，优选为15000Da至25000Da，例如15000Da至20000Da、15000Da至19000Da或17000Da至19000Da。
  15. 权利要求12-14中任一项的方法，其中所述糖胺聚糖衍生物表现出1.3-2.0的羧基增量。
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