WO2023065439A1 - 磺酸化透明质酸类化合物、其制备方法及其应用 - Google Patents

磺酸化透明质酸类化合物、其制备方法及其应用 Download PDF

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WO2023065439A1
WO2023065439A1 PCT/CN2021/130827 CN2021130827W WO2023065439A1 WO 2023065439 A1 WO2023065439 A1 WO 2023065439A1 CN 2021130827 W CN2021130827 W CN 2021130827W WO 2023065439 A1 WO2023065439 A1 WO 2023065439A1
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hyaluronic acid
fibrosis
sulfonated
acid compound
hydrogen
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French (fr)
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王春明
陈佳羲
谢达平
张哲�
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澳门大学
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    • 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
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • 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
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present disclosure relates to interdisciplinary technical fields such as medicine, material chemistry, and glycobiology, and specifically relates to sulfonated hyaluronic acid compounds, their preparation methods, and their applications.
  • Fibrosis is the main cause of disability and death in many diseases, and can occur in various organs, specifically, fibrosis and liver cirrhosis, hepatitis, nonalcoholic steatohepatitis, chronic kidney disease, myocardial infarction, heart failure, idiopathic Pulmonary fibrosis, diabetes, and scleroderma are related to many diseases, which seriously threaten human health and life.
  • TGF- ⁇ transforming growth factor- ⁇
  • ECM extracellular matrix
  • the present disclosure provides a sulfonated hyaluronic acid compound, the structural formula of which is as follows: Wherein, R is an alkali metal cation or hydrogen, R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen or sulfonate ions, and R 1 , R 2 , R 3 and R 4 cannot be hydrogen at the same time, 10 ⁇ n ⁇ 4000 and n is an integer;
  • R is sodium ion or potassium ion or hydrogen
  • R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen or sulfonate ion, and R 1 , R 2 , R 3 and R 4 cannot be hydrogen at the same time
  • R is an alkali metal cation or hydrogen
  • R 1 , R 2 , R 3 and R 4 are sulfonate ions, 2100 ⁇ n ⁇ 4000 and n is an integer.
  • the present disclosure also provides a method for preparing the above-mentioned sulfonated hyaluronic acid compound, which is carried out according to the following synthetic route:
  • the molecular weight of the raw hyaluronic acid used in the preparation method is below 1500kDa; for example, the molecular weight of the raw hyaluronic acid is any value in ⁇ 10kDa, 100-200kDa and 800kDa-1500kDa; optional Generally, the molecular weight of the raw material hyaluronic acid is 800kDa-1500kDa.
  • the sulfonating reagent used in the preparation method is pyridinic sulfur trioxide.
  • the preparation steps of the above-mentioned sulfonated hyaluronic acid compounds include: dissolving the raw material hyaluronic acid and then mixing with TBAOH for reaction, and then freeze-drying to form hyaluronic acid intermediate powder, and then, The hyaluronic acid intermediate powder is mixed with the sulfonating reagent, and the pH of the reaction system is adjusted to 8-9, followed by dialysis.
  • the present disclosure provides a hyaluronic acid nanoparticle, the structural formula of which is as follows:
  • R 1 , R 2 , R 3 and R 4 are sulfonate ions, 10 ⁇ n ⁇ 4000 and n is an integer; optionally, 2100 ⁇ n ⁇ 4000.
  • the present disclosure also provides a method for preparing hyaluronic acid nanoparticles as described above, which is carried out according to the following synthesis route:
  • the preparation method includes: ion-exchanging the sulfonated hyaluronic acid compound as described above to form a sulfonated hyaluronic acid intermediate, and then mixing with an activator, and then mixing with 4 -(1-pyrenyl)butanamide and dialyzed.
  • the activators are carbodiimide and N-hydroxysuccinimide.
  • the present disclosure also provides a glycobiological material comprising the sulfonated hyaluronic acid compound or hyaluronic acid nanoparticles as described above.
  • the present disclosure provides the use of sulfonated hyaluronic acid compounds or hyaluronic acid nanoparticles or sugar biomaterials as described above in inhibiting fibrosis or inhibiting the activation of TGF- ⁇ .
  • the present disclosure also provides a method of inhibiting fibrosis or inhibiting the activation of TGF- ⁇ in a subject in need thereof, comprising administering to the subject a sulfonated hyaluronic acid compound as described above or Hyaluronic Acid Nanoparticles or Glycobiomaterials.
  • the fibrosis comprises tissue fibrosis; alternatively, the fibrosis comprises pulmonary fibrosis, liver fibrosis, cardiac fibrosis, pancreatic fibrosis and renal fibrosis.
  • Fig. 1 is a graph showing the characterization results of the sulfonated hyaluronic acid compounds provided in Examples 1-9 of the present disclosure
  • Figure 2 is a diagram showing the NMR characterization results of 4-(1-pyrenyl)butanamide provided in Example 10 of the present disclosure
  • Figure 3 is a diagram showing the NMR characterization results of hyaluronic acid nanoparticles provided in Examples 10-12 of the present disclosure
  • Figure 4 is a particle size and morphology characterization diagram of hyaluronic acid nanoparticles provided by Examples 10-12 of the present disclosure
  • Fig. 5 is the test diagram provided by Experimental Example 1 of the present disclosure.
  • FIG. 6 is a detection chart provided by Experimental Example 2 of the present disclosure.
  • TGF- ⁇ The function of TGF- ⁇ needs to be in the form of active TGF- ⁇ .
  • the newly synthesized TGF- ⁇ forms an inactive small dormant complex with latently associated peptide (LAP) in the form of non-covalent bonds.
  • TGF-beta binding protein (LTBP) forms a large latent complex (LLC).
  • ECM extracellular matrix
  • the present disclosure provides a sulfonated hyaluronic acid compound, the structural formula of which is as follows: Wherein, R is an alkali metal cation or hydrogen, R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen or sulfonate ions, and R 1 , R 2 , R 3 and R 4 cannot be hydrogen at the same time, 10 ⁇ n ⁇ 4000 and n is an integer.
  • the sulfonated hyaluronic acid provided by the present disclosure has a strong affinity with LTBP.
  • LTBP can combine with ECM to provide sufficient mechanical force to promote TGF- ⁇ activation
  • sulfonated hyaluronic acid can be combined with LTBP has a higher affinity, thereby preventing LTBP from combining with ECM, making the mechanical force insufficient to activate TGF- ⁇ , and inhibiting fibrosis from the source of signal transduction, so it can be used to improve or treat fibrosis in subjects in need .
  • R is sodium ion or potassium ion or hydrogen
  • R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen or sulfonate ion
  • R 1 , R 2 , R 3 and R 4 cannot be hydrogen at the same time.
  • the present disclosure also provides a method for preparing the sulfonated hyaluronic acid compound described in the foregoing embodiments, and the sulfonated hyaluronic acid compound is synthesized by referring to the following synthetic route:
  • the raw material hyaluronic acid is dissolved and then mixed with tetrabutylammonium hydroxide (TBAOH) for reaction, and then freeze-dried to form hyaluronic acid intermediate powder, and then, hyaluronic acid intermediate powder and sulfonation reagent Mix and adjust the pH of the reaction system to 8-9, and then dialyze.
  • TSAOH tetrabutylammonium hydroxide
  • the above raw hyaluronic acid can be hyaluronic acid with any molecular weight, for example, the molecular weight of the raw hyaluronic acid is below 1500kDa; optionally, the molecular weight of the raw hyaluronic acid is any of ⁇ 10kDa, 100-200kDa and 800kDa-1500kDa value.
  • the sulfonating reagent used may also be an existing sulfonating reagent, for example, the sulfonating reagent is selected from pyridine sulfur trioxide.
  • the present disclosure provides a hyaluronic acid nanoparticle whose structural formula is as follows: Wherein, R 1 , R 2 , R 3 and R 4 are sulfonate ions, 10 ⁇ n ⁇ 4000 and n is an integer.
  • the present disclosure adopts the covalent combination of 4-(1-pyrenyl)butyramide and sulfonated hyaluronic acid to form hydrophilic and hydrophobic ends, which can be self-assembled to form whisker-like nanoparticles surrounded by hyaluronic acid chains, And by adjusting the amount of 4-(1-pyrenyl)butanamide, different nanoparticle sizes and compactness can be adjusted.
  • Hyaluronic acid polysaccharide chains can enrich LLC in tissues and prevent them from contacting with ECM to activate TGF- ⁇ , thereby inhibiting the activation of TGF- ⁇ .
  • the present disclosure also provides the preparation method of the hyaluronic acid nanoparticles described in the foregoing embodiments, and the hyaluronic acid nanoparticles are synthesized with reference to the following synthetic route:
  • the sulfonated hyaluronic acid compound is ion-exchanged to form a sulfonated hyaluronic acid intermediate, which is then mixed with an activator, and then mixed with 4-(1-pyrenyl)butyramide And perform dialysis.
  • the activators are carbodiimide and N-hydroxysuccinimide.
  • the present disclosure provides a glycobiological material, which includes the sulfonated hyaluronic acid compound or the hyaluronic acid nanoparticles described in the foregoing embodiment.
  • the present disclosure also provides the application of the sulfonated hyaluronic acid compound or the hyaluronic acid nanoparticles described in the foregoing embodiment or the glycobiological material described in the foregoing embodiment in inhibiting fibrosis; wherein, Fibrosis includes tissue fibrosis; for example, fibrosis includes pulmonary fibrosis, liver fibrosis, cardiac fibrosis, pancreatic fibrosis, and kidney fibrosis; the drug is a drug that inhibits the activation of TGF- ⁇ .
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method includes:
  • Low molecular weight hyaluronic acid ( ⁇ 10kDa): 10 ⁇ n ⁇ 30; Medium molecular weight hyaluronic acid: (100kDa ⁇ 200kDa): 260 ⁇ n ⁇ 530; High molecular weight hyaluronic acid: (800kDa ⁇ 1500kDa): 2100 ⁇ n ⁇ 4000.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is: pyridine sulfur trioxide—medium degree of substitution (2.28g), the raw material of hyaluronic acid is low molecular weight hyaluronic acid, and sulfonated hyaluronic acid compounds are obtained Recorded as S-HA-2.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is: pyridine sulfur trioxide - high degree of substitution (3.66g), the raw material of hyaluronic acid is low molecular weight hyaluronic acid, and sulfonated hyaluronic acid compounds are obtained Recorded as S-HA-3.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of the hyaluronic acid used is 100kDa-200kDa, and pyridine sulfur trioxide--low substitution degree, the obtained sulfonated hyaluronic acid compound is denoted as S -HA-4.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of the hyaluronic acid used is 100kDa ⁇ 200kDa, and pyridine sulfur trioxide-medium degree of substitution (2.28g), to obtain sulfonated hyaluronic acid
  • the compound is designated S-HA-5.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of the hyaluronic acid used is 100kDa ⁇ 200kDa, pyridine sulfur trioxide——high degree of substitution (3.66g), to obtain sulfonated hyaluronic acid
  • the compound is designated as S-HA-6.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of the hyaluronic acid used is high molecular weight 800kDa ⁇ 1500kDa, pyridine sulfur trioxide-low substitution degree, and the sulfonated hyaluronic acid compound is obtained. for S-HA-7.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of hyaluronic acid used is high molecular weight 800kDa ⁇ 1500kDa, and pyridine sulfur trioxide—medium degree of substitution is used to obtain sulfonated hyaluronic acid compounds.
  • the molecular weight of hyaluronic acid used is high molecular weight 800kDa ⁇ 1500kDa
  • pyridine sulfur trioxide—medium degree of substitution is used to obtain sulfonated hyaluronic acid compounds.
  • S-HA-8 For S-HA-8.
  • This example provides a sulfonated hyaluronic acid compound and a preparation method thereof.
  • Its preparation method is basically the same as the preparation method provided in Example 1, the difference is that the molecular weight of the hyaluronic acid used is high molecular weight 800kDa ⁇ 1500kDa, pyridine sulfur trioxide—high degree of substitution, and the sulfonated hyaluronic acid compound is obtained. for S-HA-9.
  • S-HA-1 to S-HA-9 were characterized by NMR technique.
  • the results are shown in Figure 1, and the analysis is as follows: the arrow mark indicates the peak shift of the methylene proton in C-6, and the triangle indicates the peak shift of the adjacent hydroxyl group, indicating S-HA-1, S-HA-2 and S-HA-3
  • S-HA-4, S-HA-5 and S-HA-6 showed a gradual increase in the degree of sulfonation substitution of HA-2 with a molecular weight of 100kDa-200kDa
  • Show that S-HA-7, S-HA-8 and S-HA-9 gradually increase the degree of sulfonation substitution of HA-3 with a molecular weight of 800kDa-1500kDa.
  • the degree of sulfonated substitution of S-HA-1 to S-HA-9 was characterized by a potentiometer, and the results are shown in Figure 1. It can be seen from the analysis that the potential of each group decreases gradually, indicating that the degree of sulfonation substitution is getting higher and higher.
  • This embodiment provides a hyaluronic acid nanoparticle and a preparation method thereof.
  • the structural formula of the hyaluronic acid nanoparticles is as follows: 2100 ⁇ n ⁇ 4000.
  • Its preparation method includes:
  • the 4-(1-pyrenyl)butyramide was characterized by NMR. The results are shown in Figure 2, and the analysis is as follows: chemical shift 8-8.5 shows obvious nine benzene ring hydrogens, and two hydrogens at chemical shift 3.5 indicate successful covalent linkage of ethylenediamine, indicating that 4-(1-pyrenyl)butyl Amide (PBA) was successfully synthesized.
  • the sulfonated hyaluronic acid compound prepared in Example 9 was passed through an ion exchange resin to replace sodium hyaluronate with sulfonated hyaluronic acid, and freeze-dried to obtain a sulfonated hyaluronic acid intermediate.
  • This embodiment provides a hyaluronic acid nanoparticle and a preparation method thereof.
  • the hyaluronic acid nanoparticles were prepared with reference to the preparation method provided in Example 10, with the difference that: the dosage of 4-(1-pyrenyl)butanamide was 50 mg, and the obtained hyaluronic acid nanoparticles were denoted as S-HA-PBA-2 .
  • This embodiment provides a hyaluronic acid nanoparticle and a preparation method thereof.
  • the hyaluronic acid nanoparticles were prepared with reference to the preparation method provided in Example 10, with the difference that: the dosage of 4-(1-pyrenyl)butyramide was 90 mg, and the obtained hyaluronic acid nanoparticles were denoted as S-HA-PBA-3 .
  • S-HA-PBA-1 to S-HA-PBA-3 were characterized by nuclear magnetic resonance and infrared spectroscopy. The results are shown in Figure 3, and the analysis is as follows: The chemical shift of 8-8.5ppm indicates the position of the benzene ring hydrogen, proving the successful covalent linkage of S-HA-PBA-1, S-HA-PBA-2 and S-HA-PBA-3 , the grafting rate is calculated by the ratio of the hydrogen of the pyrene ring to the hydrogen of the amino group, the grafting rate of S-HA-PBA-1 is 17.5%, the grafting rate of S-HA-PBA-2 is 53.2%, and the rate of S-HA-PBA-2 is 53.2%. The grafting rate of HA-PBA-3 was 86.2%.
  • the particle size and morphology of S-HA-PBA-1 to S-HA-PBA-3 were characterized by particle size analyzer and transmission electron microscope. The results are shown in Figure 4, and the analysis is as follows: Both the particle size analyzer and the transmission electron microscope showed that the nanoparticles were successfully synthesized and the particle size was about 100nm-200nm.
  • the CAGA-TGF- ⁇ activity reporter cells were plated (12-well plate) until the confluence reached 65%, and samples and reagents were added to different wells, and the grouping and concentration were as follows: The first group: 2ml TGF- ⁇ (50ng/ ml); the second group: 2ml pro-TGF- ⁇ 1 (200ng/ml); the third group: 2ml LLC (200ng/ml) + S-HA-9 (20ug/ml); the fourth group: 2ml LLC (200ng/ml) ml)+PBS(1X); the fifth group was 2ml LLC(200ng/ml)+S-HA-PBA-1(20ug/ml), stimulated overnight, collected samples, obtained cell lysate, and carried out luciferase detection.
  • the test results are shown in Figure 6. According to Figure 6, it can be seen that the sulfonated hyaluronic acid nanoparticles can inhibit the release of TGF- ⁇ . It can be seen from the figure that TGF- ⁇ stimulates to produce active TGF- ⁇ , and proTGF- ⁇ stimulates to produce a small amount of active TGF- ⁇ , indicating that the reporter cells are available, and this cell can prove the release behavior of active TGF- ⁇ from the biological experiment level, and the results are credible ;
  • the LLC+S-HA-9 group released less active TGF- ⁇ than the LLC+PBS group, indicating that S-HA-9 regulation led to insufficient mechanical force for LLC to release TGF- ⁇ , that is, inhibited the activation of TGF- ⁇ from LLC;
  • the amount of active TGF- ⁇ in the LLC+S-HA-PBA-1 group was lower than that in the LLC+S-HA-9 group, indicating that making nanoparticles is more conducive to inhibiting the release of active TGF- ⁇ .
  • the sulfonated hyaluronic acid compound of the present disclosure has a stronger interaction force with LTBP protein, thereby preventing the combination of LTBP and ECM, making the mechanical force insufficient to activate TGF- ⁇ , and realizing the inhibition of fibrosis from the source of signal transduction , has broad application prospects and high market value in the medical field involving the treatment of diseases related to fibrosis.

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Abstract

本公开涉及医学、材料化学、糖生物学等交叉技术领域，具体而言，涉及一种磺酸化透明质酸类化合物、其制备方法及其应用。该磺酸化透明质酸类化合物的结构式如式（I）所示其中，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢，10<n<4000且n为整数。该磺酸化透明质酸类化合物与LTBP蛋白具有更强的相互作用力，从而阻止LTBP和ECM结合，使得机械力不足从而无法活化TGF-β，从信号传导的源头抑制纤维化。

Description

磺酸化透明质酸类化合物、其制备方法及其应用
相关申请的交叉引用
本公开要求于2021年10月19日提交中国专利局的申请号为CN202111213457.7、名称为“磺酸化透明质酸类化合物、其制备方法及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及医学、材料化学、糖生物学等交叉技术领域，具体而言，涉及磺酸化透明质酸类化合物、其制备方法及其应用。
背景技术
纤维化是许多疾病致残、致死的主要原因，可发生于多种器官，具体地，纤维化与肝硬化、肝炎、非酒精性脂肪性肝炎、慢性肾脏病、心肌梗塞、心脏衰竭、特发性肺纤维化、糖尿病，以及硬皮病等多种疾病有关，严重威胁人类健康和生命。
截至2020年，尚无得到监管机构批准的药物可以避免或逆转纤维化过程。目前临床主要使用以代谢过程为靶点的具有部分抑制或缓解纤维化的药物为主。其中以小分子药物为主的多数抗纤维化药物都具有抗纤维化、抗炎、抗氧化作用，能够延缓纤维化导致的脏器功能下降和疾病进展，但其具体药理基础并不清楚且效果较弱。还有一些药物主要作用靶点是TGF-β-smad通路下游的蛋白及受体，其药理作用机理明确，但临床使用仅能起到改善纤维化作用或只在早期有效，且具有一定不良反应。目前研究主要集中在开发小分子药物以及在信号通路下游抑制纤维化。
在纤维化发生、发展过程中，转化生长因子-β(TGF-β)具有促进胶原蛋白基因表达、促进细胞外基质(ECM)合成与沉积等作用，是纤维化最重要的始动因子之一。TGF-β可以通过TGF-β-smad信号通路调节生理过程并发挥作用，目前大多数研究集中在对TGF-β下游信号的调控。但是少有对TGF-β活化过程的调控，在信号通路源头抑制，从而实现抗纤维化作用的研究，更没有药物能够从信号通路源头抑制，从而实现抗纤维化。
发明内容
本公开提供了一种磺酸化透明质酸类化合物，其结构式如下所示：
Figure PCTCN2021130827-appb-000001
其中，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢，10<n<4000且n为整数；
在一些实施方式中，R为钠离子或钾离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢；
在一些实施方式中，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4为磺酸盐离子，2100<n<4000且n为整数。
本公开还提供了一种如上所述的磺酸化透明质酸类化合物的制备方法，按照以下合成路径进行：
Figure PCTCN2021130827-appb-000002
在一些实施方式中，所述制备方法中所使用的原料透明质酸的分子量为1500kDa以下；例如，原料透明质酸的分子量为<10kDa、100-200kDa和800kDa-1500kDa中的任意数值；可选地，原料透明质酸的分子量为800kDa-1500kDa。
在一些实施方式中，所述制备方法中所使用的磺化试剂为吡啶三氧化硫。
在一些实施方式中，如上所述的磺酸化透明质酸类化合物的制备步骤包括：将原料透明质酸溶解后再与TBAOH混合进行反应，而后冻干形成透明质酸中间体粉末，接着，将透明质酸中间体粉末与磺化试剂混合，并将反应体系pH调至8～9，而后透析。
本公开提供了一种透明质酸纳米粒子，其结构式如下所示：
Figure PCTCN2021130827-appb-000003
其中，R 1、R 2、R 3和R 4为磺酸盐离子，10<n<4000且n为整数；可选地，2100<n<4000。
本公开还提供了一种如上所述的透明质酸纳米粒子的制备方法，按照以下合成路径进行：
Figure PCTCN2021130827-appb-000004
在一些实施方式中，所述制备方法包括：对如上文所述的磺酸化透明质酸类化合物进行离子交换形成磺酸化透明质酸中间体，而后，再与活化剂混合，接着，再与4-(1-芘基)丁酰胺混合并进行透析。
在一些实施方式中，所述活化剂是碳二亚胺和N-羟基琥珀酰亚胺。
本公开还提供了一种糖生物材料，其包括如上文所述的磺酸化透明质酸类化合物或透明质酸纳米粒子。
本公开提供了如上文所述的磺酸化透明质酸类化合物或透明质酸纳米粒子或糖生物材料在抑制纤维化或抑制TGF-β的活化中的用途。
本公开还提供了一种在有需要的受试者中抑制纤维化或抑制TGF-β的活化的方法，包括向所述受试者给药如上文所述的磺酸化透明质酸类化合物或透明质酸纳米粒子或糖生物材料。
在一些实施方式中，所述纤维化包括组织纤维化；可选地，所述纤维化包括肺纤维化、肝脏纤维化、心脏纤维化、胰腺纤维化和肾脏纤维化。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例1-9提供的磺酸化透明质酸类化合物的表征结果图；
图2为本公开实施例10提供的4-(1-芘基)丁酰胺的核磁表征结果图；
图3为本公开实施例10-12提供的透明质酸纳米粒子的核磁表征结果图；
图4为本公开实施例10-12提供的透明质酸纳米粒子的粒径和形貌表征图；
图5为本公开实验例1提供的检测图；
图6为本公开实验例2提供的检测图。
具体实施方式
下面将结合附图和实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明 具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
TGF-β发挥作用需要以活性TGF-β的形式，新合成的TGF-β以非共价键形式与潜在相关肽(LAP)形成没有活性的小休眠复合体，LAP再以二硫键与潜在TGF-β结合蛋白(LTBP)形成大潜在复合物(LLC)。TGF-β的活化需要LTBP与细胞外基质(ECM)相互作用并产生一定强度的机械力以牵引促进TGF-β脱掉“紧身衣”LAP从而成为活性TGF-β。因此，发明人研究通过调节使TGF活化过程中的机械力不足从而抑制其活化，可以减少活性TGF-β的形成，从而从根源处抑制纤维化。
鉴于此，本公开提供了一种磺酸化透明质酸类化合物，其结构式如下所示：
Figure PCTCN2021130827-appb-000005
其中，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢，10<n<4000且n为整数。
本公开提供的磺酸化的透明质酸与LTBP具有很强的亲和性，LTBP在正常生理情况下可以与ECM结合提供足够的机械力促进TGF-β活化，而磺酸化的透明质酸可以与LTBP具有更高的亲和力，从而阻止LTBP和ECM结合，使得机械力不足从而无法活化TGF-β，从信号传导的源头抑制纤维化，因而可用于改善或者治疗有需要的受试者中的纤维化。
在一些实施方式中，R为钠离子或钾离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢。
本公开还提供了前述实施方式所述的磺酸化透明质酸类化合物的制备方法，参照下述合成路径合成所述磺酸化透明质酸类化合物：
Figure PCTCN2021130827-appb-000006
在一些实施方式中，将原料透明质酸溶解后再与四丁基氢氧化铵(TBAOH)混合进行反应，而后冻干形成透明质酸中间体粉末，接着，将透明质酸中间体粉末与磺化试剂混合，并将反应体系pH调至8～9，而后透析。上述原料透明质酸可以采用任意分子量的透明质酸，例如，原料透明质酸的分子量为1500kDa以下；可选地，原料透明质酸的分子量为<10kDa、100-200kDa和800kDa-1500kDa中的任意数值。采用的磺化试剂也可以采用现有的磺化试剂，例如，磺化试剂选自吡啶三氧化硫。
此外，本公开提供了一种透明质酸纳米粒子，其结构式如下所示：
Figure PCTCN2021130827-appb-000007
其中，R 1、R 2、R 3和R 4为磺酸盐离子，10<n<4000且n为整数。在一些实施方式中，本公开采用4-(1-芘基)丁酰胺与磺酸化透明质酸共价结合形成亲疏水端，可以自主装成形成四周是透明质酸链的拂尘状纳米粒子，并且通过调节4-(1-芘基)丁酰胺的量可以调节不同的纳米粒子大小和紧实程度。透明质酸多糖链可以在组织中富集LLC并阻止其与ECM接触而活化TGF-β，起到抑制TGF-β活化的作用。
本公开还提供了前述实施方式所述的透明质酸纳米粒子的制备方法，参照下述合成路径合成透明质酸纳米粒子：
Figure PCTCN2021130827-appb-000008
在一些实施方式中，对上述磺酸化透明质酸类化合物进行离子交换形成磺酸化透明质酸中间体，而后，再与活化剂混合，接着，再与4-(1-芘基)丁酰胺混合并进行透析。可选地，活化剂为碳二亚胺和N-羟基琥珀酰亚胺。
另外，本公开提供了一种糖生物材料，其包括前述实施方式所述的磺酸化透明质酸类化合物或前述实施方式所述的透明质酸纳米粒子。
本公开还提供了前述实施方式所述的磺酸化透明质酸类化合物或前述实施方式所述的透明质酸纳米粒子或前述实施方式所述的糖生物材料在抑制纤维化中的应用；其中，纤维化包括组织纤维化；例如，纤维化包括肺纤维化、肝脏纤维化、心脏纤维化、胰腺纤维化和肾脏纤维化；药物为抑制TGF-β的活化的药物。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000009
其中，R 1＝SO 3 -，R 2＝H，R 3＝H，R 4＝H；10<n<30。
其制备方法包括：
将3g低分子量透明质酸(其分子量为<10kDa)溶于300ml去离子水中并与6ml 25％ 四丁基氢氧化铵常温搅拌反应2h，冻干得到可溶于有机试剂的透明质酸中间体粉末。取300mg透明质酸中间体粉末溶于无水二甲基甲酰胺中搅拌分散，取吡啶三氧化硫——低取代度(0.9g)溶于二甲基甲酰胺中后冰浴下加入透明质酸溶液，反应1h后加入水终止反应，用氢氧化钠调节pH 8.5，在水中透析2天，冻干得到磺酸化透明质酸类化合物，记为S-HA-1。
需要说明的是，低取代度磺酸化透明质酸：R 1＝SO 3 -，R 2＝H，R 3＝H，R 4＝H；中取代度磺酸化透明质酸：R 1＝SO 3 -，R 2＝SO 3 -，R 3＝H，R 4＝H；高取代度磺酸化透明质酸：R 1＝SO 3 -，R 2＝SO 3 -，R 3＝SO 3 -，R 4＝SO 3 -。
低分子量透明质酸：(<10kDa):10<n<30；中分子量透明质酸：(100kDa～200kDa):260<n<530；高分子量透明质酸：(800kDa～1500kDa):2100<n<4000。
实施例2
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000010
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝H，R 4＝H；10<n<30。
其制备方法与实施例1提供的制备方法基本相同，区别在于：吡啶三氧化硫——中取代度(2.28g)，透明质酸原料为低分子量透明质酸，得到磺酸化透明质酸类化合物记为S-HA-2。
实施例3
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000011
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝SO 3 -，R 4＝SO 3 -；10<n<30。
其制备方法与实施例1提供的制备方法基本相同，区别在于：吡啶三氧化硫——高取代度(3.66g)，透明质酸原料为低分子量透明质酸，得到磺酸化透明质酸类化合物记为S-HA-3。
实施例4
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000012
其中，R 1＝SO 3 -，R 2＝H，R 3＝H，R 4＝H；260<n<530。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为100kDa～200kDa，吡啶三氧化硫——低取代度，得到磺酸化透明质酸类化合物记为S-HA-4。
实施例5
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000013
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝H，R 4＝H；260<n<530。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为100kDa～200kDa，吡啶三氧化硫——中取代度(2.28g)，得到磺酸化透明质酸类化合物记为S-HA-5。
实施例6
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000014
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝SO 3 -，R 4＝SO 3 -；260<n<530。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为100kDa～200kDa，吡啶三氧化硫——高取代度(3.66g)，得到磺酸化透明质酸类化合物记为S-HA-6。
实施例7
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000015
其中， R 1＝SO 3 -，R 2＝H，R 3＝H，R 4＝H；2100<n<4000。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为高分子量800kDa～1500kDa，吡啶三氧化硫——低取代度，得到磺酸化透明质酸类化合物记为S-HA-7。
实施例8
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000016
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝H，R 4＝H；2100<n<4000。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为高分子量800kDa～1500kDa，吡啶三氧化硫——中取代度，得到磺酸化透明质酸类化合物记为S-HA-8。
实施例9
本实施例提供了一种磺酸化透明质酸类化合物及其制备方法。
该磺酸化透明质酸类化合物的结构式如下所示：
Figure PCTCN2021130827-appb-000017
其中，R 1＝SO 3 -，R 2＝SO 3 -，R 3＝SO 3 -，R 4＝SO 3 -；2100<n<4000。
其制备方法与实施例1提供的制备方法基本相同，区别在于：采用的透明质酸的分子量为高分子量800kDa～1500kDa，吡啶三氧化硫——高取代度，得到磺酸化透明质酸类化合物记为S-HA-9。
产物表征
提供了对实施例1-实施例9合成得到的磺酸化透明质酸类化合物即S-HA-1至S-HA-9进行的表征。
利用核磁共振技术对S-HA-1至S-HA-9进行表征。结果参见图1，分析如下：箭头标注表示C-6中亚甲基质子的峰移，三角形表示相邻羟基的峰移，表明S-HA-1、S-HA-2和S-HA-3比分子量<10kDa的HA-1的磺酸化取代程度逐渐增加；表明S-HA-4、S-HA-5和S-HA-6比分子量100kDa-200kDa的HA-2的磺酸化取代程度逐渐增加；表明S-HA-7、S-HA-8和S-HA-9比分子量800kDa-1500kDa的HA-3的磺酸化取代程度逐渐增加。
利用电位仪对S-HA-1至S-HA-9的磺酸化取代程度进行表征，结果参见图1。分析可知，各组电位逐渐减少表明磺酸化取代程度越来越高。
实施例10
本实施例提供了一种透明质酸纳米粒子及其制备方法。
该透明质酸纳米粒子的结构式如下所示：
Figure PCTCN2021130827-appb-000018
2100<n<4000。
其制备方法包括：
S1、制备4-(1-芘基)丁酰胺；
将500mg芘丁酸(1.73eq)溶于10ml无水二甲基甲酰胺中，加入1-[双(二甲氨基)亚甲基]-1H-1,2,3-三唑并[4,5-b]吡啶鎓3-氧化物六氟磷酸盐(HATU)1970mg(3eq)、丙二胺903mg(5.19eq)和二异丙基乙基胺(DIPEA)669mg(5.19eq)，常温搅拌反应16h，得到羧基连接乙二胺的酯。然后，用乙酸乙酯和二氯甲烷1:1萃取除去副反应产物和未反应完全的原料，并用反相硅胶色谱柱得到纯4-(1-芘基)丁酰胺。
利用核磁共振技术对4-(1-芘基)丁酰胺进行表征。结果参见图2，分析如下：化学位移8-8.5表现出明显的九个苯环氢，化学位移3.5处的两个氢表示乙二胺成功共价连接，表明4-(1-芘基)丁酰胺(PBA)成功合成。
S2、合成透明质酸纳米粒子；
将实施例9制备得到的磺酸化透明质酸类化合物通过离子交换树脂，将透明质酸钠置换成磺酸化透明质酸，冻干，得到磺酸化透明质酸中间体。将100mg得到的磺酸化透明质酸溶于10ml二甲基亚砜，溶解后加入50mg碳二亚胺和30mg的N-羟基琥珀酰亚胺，活化30min后加入60mg的4-(1-芘基)丁酰胺，超声溶解，常温搅拌16h，在水中透析2天，然后冻干得到透明质酸纳米粒子，记为S-HA-PBA-1。
实施例11
本实施例提供了一种透明质酸纳米粒子及其制备方法。参照实施例10提供的制备方法制备该透明质酸纳米粒子，区别在于：采用4-(1-芘基)丁酰胺用量为50mg，得到的透明质酸纳米粒子记为S-HA-PBA-2。
实施例12
本实施例提供了一种透明质酸纳米粒子及其制备方法。参照实施例10提供的制备方法制备该透明质酸纳米粒子，区别在于：采用4-(1-芘基)丁酰胺用量为90mg，得到的透明质酸纳米粒子记为S-HA-PBA-3。
产物表征
提供了对实施例10-实施例12制备得到的透明质酸纳米粒子即S-HA-PBA-1至S-HA-PBA-3进行的表征。
利用核磁共振技术、红外光谱技术对S-HA-PBA-1至S-HA-PBA-3进行表征。结果参见图3，分析如下：化学位移8-8.5ppm说明苯环氢的位置，证明S-HA-PBA-1、S-HA-PBA-2和S-HA-PBA-3的成功共价连接，其接枝率由芘环的氢与氨基氢的比例计算得到，S-HA-PBA-1的接枝率为17.5％，S-HA-PBA-2的接枝率为53.2％，S-HA-PBA-3的接枝率为86.2％。
通过粒度仪和透射电子显微镜对S-HA-PBA-1至S-HA-PBA-3的粒径和形貌进行表征。结果参见图4，分析如下：粒径仪和透射电子显微镜都显示纳米粒子成功合成且粒径为约100nm-200nm。
测试例1
亲和力测试
方法：制备生物素化的LTBP及分别利用实施例1-9的磺酸化透明质酸类化合物配制五个不同浓度(0.5mol/L，1mol/L，2mol/L，5mol/L，7mol/L)的待测样品，分别在生物膜干涉仪上以缓冲液60s、装载蛋白60s、缓冲液60s结合180s、解离180s的程序得到结合解离曲线，并根据分子量计算解离常数KD值，解离常数越小，亲和力越强。
结果参见图5。根据图5可知，实施例1-9的九种磺酸化程度的磺酸化透明质酸类化合物中亲和力最强的是S-HA-9。
测试例2
TGF-β活性报告细胞检测
方法：将CAGA-TGF-β活性报告细胞进行铺板(12孔板)至融合度达到65％，分别在不同孔加入样品和试剂，分组和浓度如下：第一组：2ml TGF-β(50ng/ml)；第二组2ml pro-TGF-β1(200ng/ml)；第三组：2ml LLC(200ng/ml)+S-HA-9(20ug/ml)；第四组：2ml LLC(200ng/ml)+PBS(1X)；第五组2ml LLC(200ng/ml)+S-HA-PBA-1(20ug/ml)，刺激过夜，收取样品，获得细胞裂解液，进行luciferase检测。
检测结果参见图6。根据图6可知，磺酸化透明质酸纳米粒子可以抑制TGF-β的释放。由图可知，TGF-β刺激产生活性TGF-β，proTGF-β刺激产生很少量活性TGF-β，表明报告细胞可用，该细胞可以从生物实验层面证明活性TGF-β释放行为，结果可信；LLC+S-HA-9组比LLC+PBS组释放更少的活性TGF-β，说明S-HA-9调节导致LLC释放TGF-β机械力不足，即抑制TGF-β从LLC中激活；LLC+S-HA-PBA-1组比LLC+S-HA-9组活性TGF-β量更低，说明做成纳米粒子更有利于抑制活性TGF-β释放。
以上所述仅为本公开的示例性实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开的磺酸化透明质酸类化合物与LTBP蛋白具有更强的相互作用力，从而阻止了LTBP和ECM结合，使得机械力不足从而无法活化TGF-β，实现了从信号传导的源头抑制纤维化，在涉及治疗与纤维化相关的疾病的医药领域具有广泛的应用前景和较高的市场价值。

Claims (18)

  1. 一种磺酸化透明质酸类化合物，其特征在于，其结构式如下所示：
    Figure PCTCN2021130827-appb-100001
    其中，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢，10<n<4000且n为整数。
  2. 根据权利要求1所述的磺酸化透明质酸类化合物，其特征在于，R为钠离子或钾离子或氢，R 1、R 2、R 3和R 4分别独立地选自氢或磺酸盐离子，且R 1、R 2、R 3和R 4不能同时为氢。
  3. 根据权利要求1所述的磺酸化透明质酸类化合物，其特征在于，R为碱金属阳离子或氢，R 1、R 2、R 3和R 4为磺酸盐离子，2100<n<4000且n为整数。
  4. 一种制备如权利要求1至3中任一项所述的磺酸化透明质酸类化合物的方法，其特征在于，参照下述合成路径合成所述磺酸化透明质酸类化合物：
    Figure PCTCN2021130827-appb-100002
  5. 根据权利要求4所述的方法，其特征在于，原料透明质酸的分子量为1500kDa以下；优选地，原料透明质酸的分子量为<10kDa、100-200kDa和800kDa-1500kDa中的任意数值；更优选地，原料透明质酸的分子量为800kDa-1500kDa。
  6. 根据权利要求4或5所述的方法，其特征在于，所述磺化试剂为吡啶三氧化硫。
  7. 根据权利要求4至6中任一项所述的方法，其特征在于，所述磺酸化透明质酸类化合物的制备步骤包括：将原料透明质酸溶解后再与TBAOH混合进行反应，而后冻干形成透明质酸中间体粉末，接着，将透明质酸中间体粉末与磺化试剂混合，并将反应体系pH调至8～9，而后透析。
  8. 一种透明质酸纳米粒子，其特征在于，其结构式如下所示：
    Figure PCTCN2021130827-appb-100003
    其中，R 1、R 2、R 3和R 4为磺酸盐离子，10<n<4000且n为整数；优选地，2100<n<4000。
  9. 一种制备如权利要求8所述的透明质酸纳米粒子的方法，其特征在于，参照下述合成路径合成所述透明质酸纳米粒子：
    Figure PCTCN2021130827-appb-100004
  10. 根据权利要求9所述的方法，其特征在于，包括：对如权利要求1至3中任一项所述的磺酸化透明质酸类化合物进行离子交换形成磺酸化透明质酸中间体，而后，再与活化剂混合，接着，再与4-(1-芘基)丁酰胺混合并进行透析。
  11. 根据权利要求10所述的方法，其特征在于，所述活化剂是碳二亚胺和N-羟基琥珀酰亚胺。
  12. 一种糖生物材料，其特征在于，其包括如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子。
  13. 如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料在制备抑制纤维化的药物中的用途，优选地，纤维化包括组织纤维化，更优选地，纤维化包括肺纤维化、肝脏纤维化、心脏纤维化、胰腺纤维化和肾脏纤维化。
  14. 如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料在制备抑制TGF-β的活化的药物中的用途。
  15. 如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料，用于抑制纤维化，优选地，所述纤维化包括组织纤维化，更优选地，所述纤维化包括肺纤维化、肝脏纤维化、心脏纤维化、胰腺纤维化和肾脏纤维化。
  16. 如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料，用于抑制TGF-β的活化。
  17. 一种在有需要的受试者中抑制纤维化的方法，包括：
    向所述受试者给药如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料，
    优选地，所述纤维化包括组织纤维化，更优选地，所述纤维化包括肺纤维化、肝脏纤维化、心脏纤维化、胰腺纤维化和肾脏纤维化。
  18. 一种在有需要的受试者中抑制TGF-β的活化的方法，包括：
    向所述受试者给药如权利要求1至3中任一项所述的磺酸化透明质酸类化合物或 如权利要求8所述的透明质酸纳米粒子或如权利要求12所述的糖生物材料。
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