Heparin hexasaccharide, its preparation method and application

Abstract

The application discloses a heparin hexasaccharide, a preparation method and application thereof, and a structural formula of the heparin hexasaccharide is shown in the following formula: The anticoagulant activity of HP6 is significantly lower than that of a heparin sodium standard, and the HP6 basically does not have anticoagulant activity, so that there is no risk of bleeding when the HP6 is used to exert antitumor activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a heparin hexasaccharide, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Heparin (HP) is a polysaccharide belonging to the glycosaminoglycan (GAG) family. It consists of repeating disaccharide units formed by iduronic acid (IdoA) or glucuronic acid (GlcA) linked to glucosamine (GlcN) via 1-4 glycosidic bonds. It exhibits 2-O sulfation substitution on the uronic acid residues and N-acetylation or sulfation substitution on the glucosamine residues, as well as 3-O / 6-O sulfation substitution. Recent studies have shown that the structural characteristics of HP allow it to interact with various cytokines (proteins) in vivo, exerting a series of biological functions by activating or inhibiting certain cytokines in related pathways, thus producing therapeutic or inhibitory effects on related diseases. However, due to the slight inhomogeneity of the structure of heparin and low molecular weight heparin, the various physiological activities of HP are somewhat limited. For example, its application in the treatment of tumors and other diseases may have potential side effects (such as bleeding). Furthermore, the unclear structure hinders the study of its structure-activity relationship mechanism, which is detrimental to its application beyond anticoagulation activities. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a heparin hexasaccharide, its preparation method and application, and to provide applications of HP6 in antiviral, antitumor and anti-Alzheimer's disease treatment.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] In a first aspect, the present invention provides a heparin hexasaccharide, the structural formula of which is:

[0007]

[0008] Secondly, the present invention provides a method for preparing the heparin hexasaccharide, comprising the following steps:

[0009] The heparin molecules are degraded into a multi-component oligosaccharide mixture by cleaving the glycosidic bonds between GlcNS6S(1-4)IdoA2S of heparin molecules using heparinase I.

[0010] The heparin oligosaccharide mixture was separated into oligosaccharide fragments with different molecular weight ranges using gel permeation chromatography (GPC).

[0011] Heparin hexasaccharide was prepared by separating a mixture of hexasaccharides using a strong anion exchange column (SAX).

[0012] HP6 is obtained by desalting the heparin hexasaccharide.

[0013] The heparin oligosaccharides obtained by enzymatic degradation form a carbon-carbon unsaturated double bond between C4 and C5 of the non-reducing terminal uronic acid, which can be conjugated with its carboxyl substituent and exhibit strong ultraviolet absorption at 232 nm. This provides the detection conditions for the subsequent preparation of heparin oligosaccharides.

[0014] In some embodiments, the specific operation of using heparinase I to specifically cleave the glycosidic bond between heparin molecules GlcNS6S(1-4)IdoA2S is as follows: add heparin sodium to the enzyme digestion buffer to dissolve it completely, add heparinase I, and react at 30-40°C for 3-4 hours.

[0015] Preferably, after the reaction is complete, the reaction solution is boiled to inactivate the enzyme, the supernatant is collected by centrifugation, and then freeze-dried.

[0016] In some embodiments, the specific process conditions for separating heparin oligosaccharide mixtures into oligosaccharide fragments with different molecular weight ranges using gel permeation chromatography (GPC) are as follows:

[0017] Column: GE Superdex™ Peptide 10 / 300GL gel permeation column;

[0018] Detector: UV 232nm;

[0019] Mobile phase: 0.2M ammonium bicarbonate solution (filtered through a 0.22μm membrane, vented);

[0020] Flow rate: 0.4 mL / min;

[0021] Injection volume: 120 μL.

[0022] In some embodiments, heparin hexasaccharide is prepared by separating a mixture of hexasaccharides using a strong anion exchange column (SAX), and the specific process conditions are as follows:

[0023] Chromatographic column: Waters S5 SAX sample concentration: 20 μg / μL;

[0024] Sample loading volume: 200 μL;

[0025] Flow rate: 4 mL / min;

[0026] Mobile phase: A: High-purity water, pH 3.5 (filtered and degassed);

[0027] B: 2M NaCl solution, pH 3.5 (filtered and degassed);

[0028] Detector: UV 232nm;

[0029] P max: 5MPa.

[0030] In some embodiments, the process conditions for desalting the obtained heparin hexasaccharide are as follows:

[0031] Chromatographic column: Sephadex G-10 gel chromatography column;

[0032] Sample concentration: 100 mg / mL;

[0033] Sample loading volume: 5 mL;

[0034] Flow rate: 3 mL / min;

[0035] Mobile phase: High-purity water;

[0036] Detector: UV 232nm;

[0037] P max: 200psi.

[0038] Thirdly, the present invention provides the use of the heparin hexasaccharide in the preparation of drugs for the treatment of neurodegenerative diseases and related diseases, drugs for the treatment of HIV and / or drugs for the treatment of tumors.

[0039] In some embodiments, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0040] Preferably, the relevant disease is adenovirus.

[0041] In some embodiments, the tumor is prostate cancer or liver cancer.

[0042] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0043] (1) The present invention obtains the desired heparin hexasaccharide HP6 by high performance liquid chromatography separation and purification.

[0044] (2) The heparin hexasaccharide HP6 prepared by this invention has significant biological activity, good cell affinity, and no cytotoxicity. The anticoagulant activity of HP6 is significantly lower than that of heparin sodium standard, and it basically has no anticoagulant activity. Therefore, there is no risk of bleeding when it is used to exert antitumor activity. Attached Figure Description

[0045] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0046] Figure 1 In the figure, A is a liquid chromatogram of GPC separation and preparation of crude heparin sugar in an embodiment of the present invention; B is a high performance liquid chromatogram of SAX separation and purification of heparin hexasaccharide.

[0047] Figure 2 In the text, A represents heparin hexasaccharide (HP6). 1 H spectrum; B is the heparin hexasaccharide HP6 HSQC spectrum;

[0048] Figure 3 The graph shows the cytotoxicity assay of heparin hexasaccharide HP6 in this embodiment of the invention. # indicates p≤0.05, ## indicates p≤0.01, and **** indicates p≤0.0001.

[0049] Figure 4 This is a characterization diagram of heparin hexasaccharide HP6 inhibiting TAT from penetrating the cell membrane in an embodiment of the present invention, wherein A is FITC-TAT, B is +Heparin, and C is +HP6.

[0050] Figure 5 This is a diagram illustrating the effect of the interaction between heparin hexasaccharide HP6 and Aβ on the Alzheimer's disease-related cytokine P-tau in an embodiment of the present invention.

[0051] Figure 6 In this embodiment of the invention, the inhibitory effect of heparin hexasaccharide HP6 on VIP activity is shown, where * indicates p≤0.05 and **** indicates p≤0.0001;

[0052] Figure 7 This is a comparison diagram of CD47 factor expression in lung cancer cells detected by flow cytometry in an embodiment of the present invention. ** indicates p≤0.05. Detailed Implementation

[0053] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] The present invention will be further described below with reference to the embodiments.

[0055] Example 1

[0056] The preparation method of heparin hexasaccharide is as follows:

[0057] Step (1) Preparation of oligosaccharides by heparin hydrolysis: Weigh the heparin sodium sample, add a certain amount of heparinase hydrolysis buffer, and after it is fully dissolved, add heparinase I and react in a constant temperature water bath at 40°C for 2 hours. After the reaction is completed, boil the reaction solution for 5 minutes to inactivate the enzyme, centrifuge to collect the supernatant, and freeze dry for later use.

[0058] Step (2) Lyophilization of enzymatic hydrolysis sample: Add a certain amount of high-purity water to prepare a solution, filter it through a 0.22μm filter membrane, and then separate it using a gel permeation chromatography column. Collect oligosaccharides of different molecular weights according to different elution times, lyophilize them three times to remove ammonium bicarbonate.

[0059] Step (3) The oligosaccharide structure obtained by enzymatic degradation contains unsaturated C=C double bonds and has strong absorption at 232 nm. Gel permeation chromatography was used to detect the glycosaminoglycans at 232 nm to separate the enzymatically hydrolyzed oligosaccharides, obtaining oligosaccharides of different molecular weights. The results are as follows: Figure 1 As shown in (A).

[0060] Step (4) The heparin oligosaccharide obtained by gel permeation chromatography was dissolved in high-purity water, filtered through an aqueous filter membrane, and further separated and purified by strong anion exchange chromatography to obtain the heparin hexasaccharide required for this experiment. The results are as follows: Figure 1 As shown in (B).

[0061] In step (5), the heparin oligosaccharide sample obtained by SAX separation contains non-volatile salts. In this experiment, a Sephadex G-10 column was selected to desalt the sample. The heparin oligosaccharide obtained by SAX separation was dissolved in high-purity water, filtered through a 0.22 μm aqueous phase filter membrane, and desalted using a G-10 gel chromatography column.

[0062] In step (1), the heparin sodium sample was purchased from Sanocon Biochemical Co., Ltd., with an average molecular weight of 15 kDa.

[0063] In step (2), the experimental conditions are as follows:

[0064] Instrument: Shimadzu LC-20A analytical high performance liquid chromatograph;

[0065] Column: GE Superdex™ Peptide 10 / 300GL gel permeation column;

[0066] Detector: UV 232nm;

[0067] Mobile phase: 0.2M ammonium bicarbonate solution (filtered through a 0.22μm membrane, vented);

[0068] Flow rate: 0.4 mL / min;

[0069] Injection volume: 120 μL.

[0070] In step (4), the experimental conditions are as follows:

[0071] Instrument: Shimadzu analytical high performance liquid chromatograph;

[0072] Chromatographic column: Waters S5 SAX sample concentration: 20 μg / μL;

[0073] Sample loading volume: 200 μL;

[0074] Flow rate: 4 mL / min;

[0075] Mobile phase: A: High-purity water pH=3.5 (filtered and degassed);

[0076] B: 2M NaCl solution pH=3.5 (filtered and degassed);

[0077] Detector: UV 232nm;

[0078] P max: 5MPa.

[0079] In step (5), the experimental conditions are as follows:

[0080] Instrument: Cheetah II medium-pressure preparative chromatography;

[0081] Chromatographic column: Sephadex G-10 gel chromatography column;

[0082] Sample concentration: 100 mg / mL;

[0083] Sample loading volume: 5 mL;

[0084] Flow rate: 3 mL / min;

[0085] Mobile phase: High-purity water;

[0086] Detector: UV 232nm;

[0087] P max: 200psi;

[0088] After collecting the elution peaks, the samples were combined and lyophilized.

[0089] Its NMR spectrum is shown below Figure 2 As shown in (A) and (B).

[0090] Example 2

[0091] Assay of the cytotoxicity / activity of heparin hexasaccharide HP6 on cells:

[0092] To verify whether the prepared heparin hexasaccharide is toxic to cells, the experimental steps are as follows:

[0093] Step (1) The frozen cells were thawed and placed in a 5% CO2, 37°C incubator to thaw them quickly. After thawing, the cell suspension was transferred to a centrifuge tube, culture medium was added, the cells were centrifuged, and then transferred to a culture flask and cultured in a 5% CO2, 37°C incubator.

[0094] Step (2) Cell passage: Pour out the culture medium, wash away the residual culture medium and floating dead cells with PBS, add 1 mL of 0.25% trypsin, digest at 37%, and when the cells become rounder and larger, add culture medium to stop the digestion. Use a sterile pipette to gently blow the cells to detach them from the cell wall and disperse them. Add them to a centrifuge tube, centrifuge at 1000 r / min for 3 min, discard the supernatant, blow the cell pellet with culture medium, and then evenly distribute it into two new culture flasks. Place them in a 5% CO2, 37℃ incubator for continued culture.

[0095] Step (3) When the cells have grown to about 80% of the culture flask, plate them by placing SHSY5Y cells in 96-well plates, 1×10⁶ cells per well. 5 After culturing the cells for 24 hours, the culture medium was discarded, and heparin hexasaccharide containing 1% serum was added at different concentration gradients (50-800 μg / ml). The cells were then cultured in a 5% CO2 incubator at 37°C for another 24 hours.

[0096] Step (4) MTT assay for cytotoxicity: Add 50 μl of MTT to each well containing cells, incubate at 37°C in the dark for 4 hours, then aspirate the liquid from the wells. Add 150-200 μl of DMSO to each well and continue incubation for 6 hours. After the purple product has fully dissolved, measure the absorbance at 570 nm using a microplate reader. The higher the cell proliferation rate, the higher the absorbance; the greater the cytotoxicity, the lower the absorbance. Cell viability is used as an indicator to evaluate the cytotoxic effect of the sample on cells. The results of the cytotoxicity assay are shown in [link to results]. Figure 3 The cell viability of the normal group cells decreased significantly after the addition of Aβ, but increased after the addition of HP6, demonstrating that Aβ has significant cytotoxicity to cells, while HP6 has no significant cytotoxicity to cells.

[0097] Example 3

[0098] TAT experiment and activity

[0099] To verify that heparin hexasaccharide HP6 can inhibit the transmembrane penetration of TAT protein, the experimental procedure is as follows:

[0100] Experimental drugs: HeLa cells, HP6 prepared in Example 1.

[0101] Step (1) Seed HeLa cells in 24-well plates and pre-culture them in a 5% CO2 37℃ incubator for 30 hours. After the cells have completely adhered to the plate or grown to more than 80%, discard the original culture medium and add 20 μM FITC-TAT. Incubate in a 37℃ incubator for 2 hours. Discard the culture medium and wash three times with cold PBS for two minutes each time.

[0102] After adding a certain amount of PBS in step (2), observe the cell entry of FITC-TAT under a fluorescence microscope.

[0103] The effects of adding heparin sodium in three different ways on FITC-TAT cell entry were observed. The first method involved pretreatment with heparin sodium followed by the addition of FITC-TAT; the second method involved simultaneous addition of heparin sodium and FITC-TAT; and the third method involved pretreatment of heparin sodium and FITC-TAT at room temperature in the dark before addition, with other treatments remaining the same.

[0104] Step (3) Flow cytometry was used to quantitatively study the inhibitory effect of certain glycosaminoglycans on TAT transmembrane peptides. In our experiment, two independent experiments were conducted, each containing three replicates. The efficiency of glycosaminoglycans in inhibiting TAT protein transmembrane penetration is shown in [the table below]. Figure 4 (A) Group FITC-TAT, the figure shows that the transmembrane peptide produces fluorescence in the cell; (B) Group FITC-TAT followed by Heprain, the results show that the intracellular fluorescence signal is reduced compared to group A; (C) Group HP6, the figure shows that the intracellular fluorescence signal is significantly reduced compared to groups A and B, verifying the inhibitory effect of HP6 on the transmembrane penetration of TAT transmembrane peptide.

[0105] Example 4

[0106] AD experimental details and activity

[0107] To verify that the heparin hexasaccharide HP6 prepared in Example 1 plays a regulatory role in P-tau, a key regulator of AD, the experimental procedure is as follows:

[0108] Step (1) Cell seeding: Place SHSY5Y cells in 6-well plates, 1×10⁶ cells per well. 6 Each cell.

[0109] Step (2) Select 10-50 μM Aβ and 50-150 μg / ml HP6 and incubate under the same conditions, and set up a negative control group (only add medium containing 1% FBS).

[0110] Step (3) 24 hours later, protein was extracted as a Western blot sample. The sample was loaded according to the specified ratio, and electrophoresis was performed. Electrophoresis was stopped when the sample reached the lowest point. After gel transfer, the sample was incubated with primary and secondary antibodies sequentially. After washing, developing solution was added, and the sample was subjected to chemiluminescence imaging using a gel imaging system to obtain the expression levels of P-tau factor in cells under different treatment conditions. Chemiluminescence imaging using a gel imaging system was used to obtain... Figure 5As shown in the figure, the expression level of P-tau in the groups with added Aβ was significantly increased compared to the normal group, proving that Aβ can induce an increase in intracellular P-tau expression. The enrichment of P-tau is considered to be driven by amyloid protein and has an indicative role in AD pathology.

[0111] Example 5

[0112] Tumor Experiment Details and Activity

[0113] Experimental Example 1: Effects of Heparin Hexasaccharide (HP6) and VIP on cAMP

[0114] To verify that heparin hexasaccharide can regulate cAMP-related pathways by modulating VIP protein, the experimental procedure is as follows:

[0115] Step (1) Cell resuscitation and passage: Wash away residual culture medium and floating dead cells with PBS, add 1 mL of 0.25% EDTA-trypsin, digest in a 37℃ incubator, and when the cells become round and larger, add serum-containing culture medium to stop digestion. Gently pipette the cells to detach them from the cell wall and disperse them, add them to a centrifuge tube, centrifuge at 5000 r / min for 3 min, discard the supernatant, pipette the cell pellet with culture medium, and then evenly distribute it into two new culture flasks and incubate in a 5% CO2, 37℃ incubator.

[0116] Step (2) Effect of VIP on cAMP concentration in PC3 cells: PC3 cells were placed in 12-well plates, with 1 × 10⁶ cells per well. 6 After culturing cells for 24-36 hours, the culture medium was discarded. 0-0.5 μM HP6 and a positive control group (VIP antagonist) were pre-incubated at room temperature for 0-30 minutes before being added. A negative control group was also included (containing only 0-0.4 μM VIP). The immunofluorescence patterns are shown below. Figure 6 The figure shows the statistical analysis of the inhibition rate of HP6 on VIP activity at different concentrations. As can be seen from the figure, the inhibitory effect on VIP increases with the increase of HP6 concentration.

[0117] Experimental Example 2: Effects of Heparin Hexasaccharide (HP6) and IFN on Lung Cancer

[0118] To verify the regulatory effect of heparin hexasaccharide on IFN, a key factor in lung cancer, the experimental procedure is as follows:

[0119] Experimental drug: Heparin hexasaccharide HP6 prepared in Example 1.

[0120] Experimental methods:

[0121] Step (1) Cell thawing: Place the frozen A549 cells in a 37°C water bath to thaw them quickly. After thawing, transfer the cell suspension to a centrifuge tube, add culture medium, centrifuge, and then transfer the suspension to a culture flask. Place the flask in a 5% CO2, 37°C incubator for culture.

[0122] Step (2) Passage the cells when they reach the fusion state.

[0123] Step (3) Cell culture and passage: Pour out the culture medium, wash away the residual culture medium and floating dead cells with PBS, add 1 mL of 0.25% trypsin, digest at 37%, and when the cells become rounder and larger, add culture medium to stop digestion. Use a sterile pipette to gently blow the cells to detach them from the cell wall and disperse them. Add them to a centrifuge tube, centrifuge at 1000 r / min for 3 min, discard the supernatant, blow the cell pellet with culture medium, and then evenly distribute it into two new culture flasks and place them in a 5% CO2, 37℃ incubator for culture.

[0124] Step (4) Flow cytometry detection of APC channel fluorescence expression changes: A549 cells were placed in 12-well plates and cultured for 0-36 h. After culturing, the culture medium was discarded, and 1 ml of PBS was slowly added along the sidewall for washing once. Then, 1 ml of HP6 was added in the form of a medium change, and the cells were cultured in an incubator. After incubation for 24-36 h, the culture medium was discarded, and 1 ml of PBS was added for washing once. The PBS was discarded, and 500 μl of trypsin was added to each well for digestion. After 3 min, 1 ml of culture medium was added to stop the digestion and the cells were pipetted. The cell suspension was then transferred to 2 ml EP tubes, centrifuged to remove the supernatant, and the cells were collected. 1 ml of PBS was added, the cells were pipetted and counted, and the solution was diluted to 1 × 10⁶ cells per well. 7 Each cell.

[0125] Step (5) Transfer 100 μl of cell suspension to a 1.5 ml EP tube, add 3-6 ml of CD47-APC antibody to each tube, and incubate at 4°C in the dark for 45-50 min. After incubation, add 1 ml of PBS, mix well, and centrifuge to remove the supernatant. Finally, add 400 μl of PBS, mix well, and prepare the cell suspension for testing. Detect the fluorescence expression using flow cytometry. Figure 7 As shown, the expression level of CD47 decreased significantly after the addition of HP6, indicating that HP6 can inhibit the expression of CD47.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a heparin hexasaccharide in the preparation of anti-Alzheimer's disease therapeutic drugs and anti-HIV therapeutic drugs, characterized in that: The preparation method of the heparin hexasaccharide includes the following steps: The heparin molecules are degraded into a multi-component oligosaccharide mixture by cleaving the glycosidic bonds between GlcNS6S(1-4)IdoA2S of heparin molecules using heparinase I. Heparin oligosaccharide mixtures were separated into oligosaccharide fragments with different molecular weight ranges using a gel permeation chromatography column; Heparin hexasaccharide was prepared by separating a mixture of hexasaccharides using a strong anion exchange column. Desalting the obtained heparin hexasaccharide yields HP6; The structural formula of the heparin hexasaccharide is: 。 2. The application according to claim 1, characterized in that: The specific procedure for using heparinase I to specifically cleave the glycosidic bond between heparin molecules GlcNS6S(1-4)IdoA2S is as follows: Add heparin sodium to the enzyme digestion buffer to dissolve it completely, add heparinase I, and react at 30-40℃ for 3-4 hours.

3. The application according to claim 1, characterized in that: After the reaction is complete, boil the reaction solution to deactivate the enzyme, centrifuge to collect the supernatant, and freeze-dry.

4. The application according to claim 1, characterized in that: The specific process conditions for separating heparin oligosaccharide mixtures into oligosaccharide fragments with different molecular weight ranges using gel permeation chromatography are as follows: Column: GE Superdex™ Peptide 10 / 300 GL gel permeation column; Detector: UV 232 nm; Mobile phase: 0.2 M ammonium bicarbonate solution; Flow rate: 0.4 mL / min; Injection volume: 120 μL.

5. The application according to claim 1, characterized in that: Heparin hexasaccharide was prepared by separating a mixture of hexasaccharides using a strong anion exchange column, and the specific process conditions were as follows: Column: Waters Spherisorb® S5 SAX; Sample concentration: 20 μg / μL; Sample loading volume: 200 μL; Flow rate: 4 mL / min; Mobile phase: A: High-purity water, pH 3.5; B: 2 M NaCl solution, pH 3.5; Detector: UV 232 nm; P max: 5 MPa.

6. The application according to claim 1, characterized in that: The desalting process conditions for the obtained heparin hexasaccharide are as follows: Chromatographic column: Sephadex G-10 gel chromatography column; Sample concentration: 100 mg / mL; Sample loading volume: 5 mL; Flow rate: 3 mL / min; Mobile phase: High-purity water; Detector: UV 232 nm; P max: 200 psi.

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