CN114591451A

CN114591451A - Partial anticoagulant heparin derivative and preparation method and application thereof

Info

Publication number: CN114591451A
Application number: CN202210176221.9A
Authority: CN
Inventors: 张真庆; 陈磊; 欧阳艺兰; 易琳; 李笃信
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-06-07

Abstract

The invention discloses a partial anticoagulation heparin derivative, wherein the ring opening rate of 2, 3-position uronic acid is 2-12%, the molecular weight is 14-18 kDa, the anti-Xa factor is more than or equal to 80IU/mg and less than 150IU/mg, and the preparation method comprises the following steps: adding sodium periodate into heparin sodium serving as a raw material to perform partial oxidation; adding sodium borohydride for reduction; and (4) removing salt by membrane filtration. The partially anticoagulated heparin disclosed by the invention has anti-inflammatory and anticoagulation effects, but the anticoagulation effect with the same quality is lower than that of heparin sodium, so that the bleeding risk of the heparin sodium in clinical use is reduced, and the partially anticoagulated heparin is safer and more controllable; the potency is stable, the structure is clear; the method uses the refined heparin as a raw material, has simple process and is suitable for large-scale production.

Description

Partial anticoagulant heparin derivative and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a partially anticoagulant heparin derivative, and a preparation method and application thereof.

Background

Heparin (Heparin) is a glycosaminoglycan that has been used clinically for over 80 years as an anticoagulant drug. Heparin, mainly derived from the porcine small intestine and bovine lung, is a polysulfated linear polysaccharide consisting of uronic acid and glucosamine as the smallest disaccharide repeating unit. The anticoagulant activity of the pentasaccharide fragment is related to the specific binding of 3-O sulfated pentasaccharide fragment GlcNAc/NS6S (1-4) GlcA (1-4) GlcNS3S,6S (1-4) IdoA2S (1-4) GlcNS6S to antithrombin. Heparin has been shown to have, in addition to its anticoagulant effect, non-anticoagulant activity such as anti-inflammatory, anti-tumor, anti-viral and neuroprotective activity (Li J P, Vlodavsky I. heparin, heparin sulfate and heparin in antibiotic reactions, Thrombosins & Haemostatis, 2009,102(11): 823-828.).

Sepsis is an acute disease with systemic inflammatory reactions and thrombosis caused by infection and aggravated sepsis (Levi M, Tom v. diagnosis and sepsis. thrombosis Research,2017,149(Complete): 38-44.). Heparin, when considering its simultaneous anticoagulant and anti-inflammatory activity, is a potential therapeutic agent for sepsis. However, the strong anticoagulation effect of heparin increases the generation of bleeding side effects, and limits the dosage of the drug. Therefore, in order to reduce the bleeding risk caused by the strong anticoagulation activity of heparin, there are patents or documents in which the anticoagulation structure site is completely destroyed by modifying the heparin structure by chemical means, so as to obtain the anticoagulated heparin derivative. Chinese patent document CN108424474A (application No. 201710815567.8) discloses a anticoagulated open-loop heparin derivative with anti-Xa factor less than or equal to 70 IU/mg. However, sepsis is characterized by the occurrence of systemic inflammatory reaction accompanied by the occurrence of thrombus, and completely open-loop anticoagulated heparin cannot satisfy thrombus treatment for patients with sepsis.

Therefore, there is a need to develop partially anticoagulant heparin derivatives, and methods for preparing and using the same to solve the above problems.

Disclosure of Invention

The invention aims to provide a partial anticoagulation heparin derivative, a preparation method and application thereof.

The invention has a technical scheme that:

a partially anticoagulant heparin derivative comprising the following general structure:

wherein the content of the first and second substances,

R₁is any one of hydrogen, acetyl or sulfonic group;

R₂is any one of hydrogen or sulfonic acid group;

R₃is any one of hydrogen, acetyl or sulfonic group;

R₄is any one of hydrogen or sulfonic acid group;

R₅is any one of hydrogen or sulfonic acid group;

n is any natural number between 1 and 40, m is any natural number between 0 and 10, and m + n is greater than or equal to 10 and less than or equal to 40.

Furthermore, the opening rate of the

uronic acid

2,3 position of the partially anticoagulated heparin derivative is 2-12%, the molecular weight is 14 kDa-18 kDa, and the anti-Xa factor of the partially anticoagulated heparin derivative is greater than or equal to 80IU/mg and less than 150 IU/mg.

Further, the partial anticoagulation heparin derivatives are PACH-1, PACH-3 and PACH-6.

The other technical scheme of the invention is as follows:

a process for the preparation of a partially anticoagulant heparin derivative, comprising the steps of:

1) adding sodium periodate into heparin sodium serving as a raw material to perform partial oxidation;

2) adding sodium borohydride for reduction;

3) and (4) removing salt by membrane filtration.

Further, in the step 1), the mass ratio of sodium heparin to sodium periodate is 10: 1-5, dissolving heparin sodium in ultrapure water, wherein the material-water ratio of the heparin sodium to the ultrapure water is 10 g: 50-1000 mL.

Further, in step 1), the partial oxidation is specifically: adjusting the pH value to 4.0-5.0, and carrying out partial oxidation for 24 hours in a dark place at the temperature of 4 ℃.

Further, in the step 2), the mass ratio of the sodium borohydride to the sodium periodate is 1-5: 1 to 5.

Further, before step 3), the pH was first adjusted to 7.0.

Further, in the step 3), the membrane filtration is any one of dialysis, ultrafiltration and tangential flow filtration, and when ultrafiltration is selected, the pore size of the membrane is 0.5kDa to 2 kDa.

The application of the partially anticoagulated heparin derivative prepared by the method in the preparation of anticoagulation and/or anti-inflammatory active medicaments.

The invention provides a partial anticoagulation heparin derivative, a preparation method and an application thereof, and the partial anticoagulation heparin (PACH) with partial anticoagulation activity is particularly important, and the anti-Xa factor is more than or equal to 80IU/mg and less than 150 IU/mg). The method has the following specific advantages:

1) has anti-inflammatory function superior to that of refined heparin raw material;

2) partial anticoagulation titer is kept, bleeding risk accompanied by heparin in clinical use is reduced, and the heparin is safer and more controllable;

3) the potency is stable, the structure is clear;

4) the refined heparin is used as a raw material, the process is simple, and the method is suitable for large-scale production.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,

FIG. 1 is a graph showing the molecular weight distribution of partially anticoagulated heparin (PACH) and heparin in a partially anticoagulated heparin derivative according to the present invention;

FIG. 2 is a graph showing the sulfate content distribution of partially anticoagulated heparin (PACH) and heparin bulk drug in a partially anticoagulated heparin derivative according to the present invention;

FIG. 3 is a one-dimensional SAX UV chromatogram of a partially anticoagulated heparin derivative (PACH) and heparin drug after heparinase degradation and 2D-LC-MS analysis according to the present invention;

FIG. 4 is a graph showing the anti-Xa factor activity profile of partially anticoagulated heparin (PACH) and heparin prodrugs of a partially anticoagulated heparin derivative according to the present invention;

FIG. 5 is a graph showing the relationship between partial anticoagulated heparin (PACH) and heparin drug in the partial anticoagulated heparin derivative according to the present invention and the protective effect of LPS-induced RAW264.7 cell inflammation model (NO content level);

FIG. 6 is a graph showing the relationship between partial anticoagulation heparin (PACH) and heparin drug in LPS-induced BMDM cell inflammation model (iNOS expression level) in a partially anticoagulation heparin derivative according to the present invention.

Detailed Description

The invention discloses a partial anticoagulation heparin derivative (PACH) with anti-inflammatory activity, wherein a2, 3-bit vicinal diol structure contained in partial uronic acid in a partial anticoagulation heparin structure is subjected to characteristic oxidative ring opening to obtain a2, 3-bit open ring uronic acid structure, the 2, 3-bit open ring ratio of the uronic acid is 2-12%, preferably less than or equal to 6%, the molecular weight is 14-18 kDa, preferably 15kDa, and the anti-Xa factor of the partial anticoagulation heparin is more than or equal to 80IU/mg and less than 150 IU/mg. The structure is as follows:

wherein R is₁、R₃Can be hydrogen, acetyl or sulfonic; r₂、R₄And R₅Can be hydrogen or sulfonic acid group, n is any natural number between 1 and 40, m is any natural number between 0 and 10, and m + n is greater than or equal to 10 and less than or equal to 40.

The preparation process of the structure comprises the following steps:

1) dissolving heparin sodium serving as a raw material in ultrapure water, adding sodium periodate, adjusting the pH value to 4.0-5.0, and performing partial oxidation for 24 hours in a dark condition at the temperature of 4 ℃, wherein the mass ratio of the heparin sodium to the sodium periodate is 10: 1-5, wherein the material-water ratio of the heparin sodium to the ultrapure water is 10 g: 50-1000 mL;

2) adding sodium borohydride for reduction, wherein the mass ratio of the sodium borohydride to the sodium periodate is 1-5: 1-5;

3) adjusting the pH value to 7.0, and desalting by membrane filtration, wherein the desalting by membrane filtration can be dialysis, ultrafiltration, tangential flow filtration and the like, and ultrafiltration is preferred; the membrane aperture is 0.5kDa to 2kDa, preferably 1 kDa;

the partial anticoagulant active heparin PACH prepared by taking heparin sodium as a raw material has both anti-inflammatory and anticoagulant effects, the anti-inflammatory activity of the partial anticoagulant active heparin PACH is superior to that of a refined heparin raw material, and meanwhile, the reserved partial anticoagulant potency effectively reduces the bleeding side effect of the heparin raw material, so that the partial anticoagulant active heparin PACH has great application potential to diseases of inflammation and blood coagulation interweaving such as septicemia and sepsis.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.

First, the references herein to "one embodiment" or "an embodiment" refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual manufacturing process should include three-dimensional space of length, width and depth.

Example 1

Preparation of partially anticoagulated heparin (PACH)

Using the finished product of heparin sodium, respectively weighing 1g of the finished product and dissolving the weighed raw materials in 3 100mL conical flasks according to the material-water ratio of 10:1(g: L). Sodium periodate was added as an oxidizing agent to sodium heparin at 10:1, 8:1 and 5:1(g: g), respectively, and the pH was adjusted to 4.0 with hydrochloric acid. The reaction was carried out at 4 ℃ for 24 hours under protection from light. Using a rubber head dropper to transfer 10mL of glycol to terminate the reaction, adopting a roll type organic membrane with the pore diameter of 1000Da to carry out desalination treatment, changing water every 1h, and dialyzing for 24 h. The dialysate was concentrated to 100mL using 250mL eggplant-shaped rotary evaporation bottles and transferred to 100mL conical flasks. Sodium borohydride (200 mg) was added thereto, and the mixture was reacted at room temperature in the dark for 4 hours with stirring. Adjusting pH to 7.0 with diluted NaOH solution to stop reaction, desalting with roll-type organic membrane with pore diameter of 1000Da, changing water every 1 hr, and dialyzing for 24 hr. The dialysate was then concentrated using a 250mL eggplant-shaped rotary evaporator. After freeze drying, 865.3mg of PACH-1, 841.9mg of PACH-2 and 822.1mg of PACH-3 are obtained in turn.

Example 2

Preparation of partially anticoagulated heparin (PACH)

Using the finished product of heparin sodium, respectively weighing 1g of the finished product and dissolving the weighed raw materials in 3 100mL conical flasks according to the material-water ratio of 10:1(g: L). Sodium periodate was added as an oxidizing agent to sodium heparin at ratios of 10:1, 4:1 and 1:1(g: g), respectively, and the pH was adjusted to 4.0 with hydrochloric acid. The reaction was carried out at 4 ℃ for 24 hours under protection from light. Using a rubber head dropper to transfer 10mL of glycol to terminate the reaction, adopting a roll type organic membrane with the pore diameter of 1000Da to carry out desalination treatment, changing water every 1h, and dialyzing for 24 h. The dialysate was concentrated to 100mL using 250mL eggplant-shaped rotary evaporation bottles and transferred to 100mL conical flasks. Sodium borohydride (200 mg) was added thereto, and the mixture was reacted at room temperature in the dark for 4 hours with stirring. Adjusting pH to 7.0 with diluted NaOH solution to stop reaction, desalting with roll-type organic membrane with pore diameter of 1000Da, changing water every 1 hr, and dialyzing for 24 hr. The dialysate was then concentrated using a 250mL eggplant-shaped rotary evaporator. After freeze drying, the partially anticoagulant heparin 883.9mg PACH-4, 854.6mg PACH-5 and 793.0mg PACH-6 are obtained in turn

Structural characterization of partially anticoagulated heparin (PACH)

And selecting a refined heparin sodium raw material drug (150.1IU/mg) and the partially anticoagulated heparin PACH-1(133.5IU/mg), PACH-3(117.0IU/mg) and PACH-6(88.0IU/mg) obtained in the examples 1 and 2 to perform qualitative and quantitative analysis on molecular weight, sulfate radical content and two-dimensional liquid phase-mass spectrum respectively.

(1) Molecular weight analysis

About 10mg of sodium heparin, PACH-1, PACH-3 and PACH-6 in examples 1 and 2 were precisely weighed, dissolved in ultrapure water, to prepare a sample solution having a concentration of 10mg/mL, and filtered through a 0.22 μm filter membrane. The molecular weight of dextran reference substance, sodium heparin, PACH-1, PACH-3 and PACH-6 was measured by size exclusion chromatography combined with laser scattering detection (SEC-MALS), please refer to FIG. 1, FIG. 1 is a graph showing the molecular weight distribution of partially anticoagulated heparin (PACH) and heparin bulk drug in partially anticoagulated heparin derivative according to the present invention. As shown in FIG. 1, the dn/dc value was 0.1380mL/g, and the relative molecular weights of PACH-1, PACH-3 and PACH-6 were: 16.6kDa, 15.0kDa, 14.3kDa and 14.0kDa, and from the data results, the molecular weight of PACH prepared from heparin is not greatly changed, and a macromolecular long-chain structure is reserved.

(2) Analysis of sulfate radical content

In examples 1 and 2, about 15mg of sodium heparin, PACH-1, PACH-3 and PACH-6 were precisely weighed and dissolved in 2M trifluoroacetic acid (TFA) to prepare a 5mg/mL solution. Heating and degrading for 24h at 100 ℃. And (3) taking 100 mu L of degraded sample, carrying out rotary evaporation to remove TFA in the sample, fixing the volume by using a 20mL volumetric flask, taking 2mL of sample solution with fixed volume, diluting by 5 times by using purified water, finally diluting to the sample loading concentration of 5ppm, and filtering by using a 0.22 mu m water system filter membrane. Referring to fig. 2, fig. 2 is a graph showing the sulfate content distribution of partial anticoagulant heparin (PACH) and heparin bulk drug in a partial anticoagulant heparin derivative according to the present invention. As shown in figure 2, the degraded sample and the standard solution are separated and analyzed by ion chromatography, and the sulfate radical contents (mass percentage) of PACH-1, PACH-3 and PACH-6 are as follows in sequence: 29.3%, 30.2%, 29.7%, 31.5%, determining the content of partial anticoagulant heparin sulfate radical obtained by preparation is about 30%, which is basically consistent with heparin original drug.

(3) Characteristic structure, open loop ratio analysis

About 1mg of sodium heparin, PACH-1, PACH-3 and PACH-6 in examples 1 and 2 were precisely weighed and dissolved in pure water to prepare 5mg/mL solutions. Respectively taking 50 mu L of the anticoagulant heparin derivative, adding 0.1IU of each of heparinases III and III, degrading for 24 hours at 37 ℃, filtering by a water system filter membrane with the thickness of 0.22 mu m, and analyzing by liquid chromatography mass spectrometry (HPLC-QTOFMS), as shown in figure 3, wherein figure 3 is a one-dimensional SAX ultraviolet chromatogram of a part of anticoagulant heparin derivative (PACH) and a heparin raw drug of the invention after heparinases degradation and 2D-LC-MS analysis. As shown in FIG. 3, as the degree of oxidation increases from heparin to PACH1, PACH3, and PACH6, the content of the ortho-dihydroxy structure formed after ring opening increases, and the ring opening ratio increases. Table 1 shows the composition of the peaks of the heparins of the invention after heparinase degradation and 2D-LC-MS analysis, and the corresponding oligosaccharide sequence table, using the standard nomenclature [ Δ HexA; HexA; GlcN; ac; SO (SO)₃；gsHexA]The composition of each chromatogram peak is shown, wherein gsHexA represents open ring uronic acid, the numbers in brackets represent the number of each oligosaccharide building block, and the structural sequence is shown in Table 1.

TABLE 1

As can be seen from Table 1, by periodic acid partial oxidation and sodium borohydride reduction, a part of uronic acid containing 2, 3-vicinal diol in the heparin sugar chain is characteristically oxidatively ring-opened, and ring-opened heparin is obtained. The open loop rate of each sample can be calculated according to the quantitative and qualitative analysis of each peak, and the oxidation degree of each sample can be characterized. The calculation formula is as follows:

wherein S is the chromatographic peak area.

Through calculation, the ring opening rates of the heparin sodium, the PACH-1, the PACH-3 and the PACH-6 are as follows in sequence: 0%, 4.1%, 7.6% and 11.0%.

Example 3

Determination of anti-factor Xa Activity of partially anticoagulated heparin (PACH)

The sodium heparin original drug, PACH-1, PACH-2, PACH-3, PACH-4, PACH-5 and PACH-6 in the examples 1 and 2 are respectively dissolved in ultrapure water to prepare 2mg/mL solution, and the solution is gradually diluted to the concentration to be measured of 0.1 mu g/mL. Heparin series standards were diluted and configured to gradient concentrations of 0IU/mL, 0.0073IU/mL, 0.0096IU/mL, 0.0143IU/mL, and 0.0189IU/mL for the plotting of anti-FXa standard curves. Three parallel samples are respectively arranged on all samples to be detected and each concentration heparin standard in the standard curve. Adding 40 mu L of each sample and heparin standard substance with serial concentrations into a 96-well plate to be detected; then adding Reagent 1(AT III) AT 37 ℃ and oscillating for 2min on a constant temperature oscillator; immediately adding Reagent 2(FXa) and accurately oscillating for 2 min; adding Reagent3(Substrate) and shaking for 2 min; the reaction was terminated by adding 2% aqueous citric acid solution, and the absorbance was measured at 405 nm. Referring to FIG. 4, FIG. 4 is a graph showing the anti-Xa factor activity of partially anticoagulated heparin (PACH) and heparin as a raw heparin in a partially anticoagulated heparin derivative according to the present invention. As shown in fig. 4, a standard curve of FXa resistance was plotted based on the absorbance values of the standard solutions of heparin-series gradient concentrations, and the absorbance values of the control and the sample were substituted into the standard curve to calculate the FXa resistance titer. The results show that the anticoagulant potency of heparin sodium, PACH-1, PACH-2, PACH-3, PACH-4, PACH-5 and PACH-6 are as follows in sequence: 150.1IU/mg, 133.5IU/mg, 123.7IU/mg, 117.0IU/mg, 128.7IU/mg, 94.7IU/mg and 88.0 IU/mg.

Evaluation of anti-inflammatory Activity of partially anticoagulated heparin (PACH) (1)

Sodium heparin, PACH-1, PACH-3 and PACH-6 in examples 1 and 2 were precisely weighed at about 5mg, and each of them was dissolved in PBS to prepare a 5mg/mL solution, which was then filtered through a 0.22 μm filterAnd (5) filtering and sterilizing. The RAW264.7 cells frozen at-80 ℃ were removed and rapidly transferred to a 37 ℃ water bath, and when the cells were to be completely thawed, they were transferred in a clean bench to a 15mL centrifuge tube containing 4mL of cell culture medium and centrifuged (800r/min 5 min). Discarding the supernatant, adding 1mL of cell culture medium, gently blowing the pellet slowly to resuspend the cells, resuspending the cells, transferring the resuspended cells to a cell culture dish, and placing the dish at 37 deg.C and 5% CO₂The incubator of (2) for cultivation. And observing the growth condition of the cells after 24h, replacing a fresh cell culture solution if the adherent growth of the cells is good, and observing the growth condition of the cells after continuously culturing for 24 h. This procedure was repeated until the cell density reached 70-80%, the cells were scraped off using a cell scraper, diluted to a density of about 20X 104 cells/mL with DMEM medium, seeded into 96-well plates at 100. mu.L/well, and placed at 37 ℃ in a 5% CO medium₂Culturing in an incubator for 12 h. The sample was added to a final concentration of 500. mu.g/mL for pre-protection for 12 h. Then adding LPS solution with the mass concentration of 100 ng/mL. After 12 hours of culture, the cell supernatants in the 96-well plate were measured using a Nitric Oxide (NO) assay kit, and the results are shown in fig. 5, and fig. 5 is a graph showing the protective effect of partial anticoagulation heparin (PACH) and heparin prodrugs on LPS-induced RAW264.7 cell inflammation model (NO content level) in a partial anticoagulation heparin derivative according to the present invention. As shown in FIG. 5, heparin sodium, PACH-1, PACH-3 and PACH-6 can significantly reduce NO levels and have good protective effect on inflammation models induced by LPS. Wherein, partial anticoagulation heparin PACH-1 has the most outstanding anti-inflammatory capability and is superior to the heparin sodium raw material.

Evaluation of anti-inflammatory Activity of partially anticoagulated heparin (PACH) (2)

Sodium heparin, PACH-1, PACH-3 and PACH-6 in examples 1 and 2 were precisely weighed at about 5mg, and each of them was dissolved in PBS to prepare a 5mg/mL solution, which was sterilized by filtration through a 0.22 μm filter. A Balb/c mouse is taken, after euthanasia, the hind leg tibia of the mouse is taken, the muscle is removed, a Running Buffer is sucked by a 5mL syringe, the bone marrow is blown out to a centrifuge tube, then the centrifuge is carried out at 300rpm for 5min, and the supernatant is discarded. The erythrocytes were lysed with 1X erythrocyte lysate for 6min, followed by addition of Running Buffer for discontinuation, centrifugation at 300rpm for 5min, and the supernatant discarded. After cell counting, the cells were plated in 6-well plates in a fixed number of cells and supplemented with Murine macrophage colony stimulating factor (Murine M-CSF) (20 ng/mL). After four days, the culture medium was replaced and Murine M-CSF was added. After the liquid is changed, the BMDM can be harvested for standby after two days. M1 classical stimulation (LPS 100ng/mL) is added into BMDM to induce typing, experimental drugs are added at the same time, after 12h of culture, RNA is collected from cells, and real-time-PCR is carried out after cDNA inversion to detect the expression condition of iNOS genes. Referring to fig. 6, fig. 6 is a graph showing the relationship between partial anticoagulation heparin (PACH) and heparin drug substance in the partially anticoagulation heparin derivative according to the present invention and the protection effect of the BMDM cell inflammation model induced by LPS (iNOS expression level). As shown in FIG. 6, the gene expression of iNOS was significantly reduced by heparin, PACH-1, PACH-3, and PACH-6, in which PACH-1 had the most significant effect and was superior to the refined heparin sodium technical

Compared with the prior art, the invention has the beneficial effects that: the invention provides a partial anticoagulation heparin derivative, a preparation method and application thereof, which show stronger anti-inflammatory action while retaining certain anticoagulation activity, are fit for diseases of septicemia, sepsis and other inflammations and coagulation interweaving, and have wide application space.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A partially anticoagulant heparin derivative is characterized by comprising the following general structure:

wherein the content of the first and second substances,

R₁is any one of hydrogen, acetyl or sulfonic group;

R₂is any one of hydrogen or sulfonic acid group;

R₃is any one of hydrogen, acetyl or sulfonic group;

R₄is any one of hydrogen or sulfonic acid group;

R₅is any one of hydrogen or sulfonic acid group;

2. A partially anticoagulant heparin derivative according to claim 1, wherein: the opening rate of the uronic acid 2,3 position of the partial anticoagulation heparin derivative is 2-12%, the molecular weight is 14-18 kDa, and the anti-Xa factor of the partial anticoagulation heparin derivative is more than or equal to 80IU/mg and less than 150 IU/mg.

3. A partially anticoagulant heparin derivative according to claim 1, wherein: the partial anticoagulation heparin derivatives are PACH-1, PACH-3 and PACH-6.

4. A method for preparing a partially anticoagulant heparin derivative according to any one of claims 1 to 3, comprising the steps of:

2) adding sodium borohydride for reduction;

3) and (4) removing salt by membrane filtration.

5. The process for the preparation of partially anticoagulant heparin derivatives according to claim 4, wherein: in the step 1), the mass ratio of sodium heparin to sodium periodate is 10: 1-5, dissolving heparin sodium in ultrapure water, wherein the material-water ratio of the heparin sodium to the ultrapure water is 10 g: 50-1000 mL.

6. The process for the preparation of partially anticoagulant heparin derivatives according to claim 5, wherein, in step 1), the partial oxidation is specifically: adjusting the pH value to 4.0-5.0, and carrying out partial oxidation for 24 hours in a dark place at the temperature of 4 ℃.

7. The process for the preparation of partially anticoagulant heparin derivatives according to claim 5, wherein: in the step 2), the mass ratio of the sodium borohydride to the sodium periodate is 1-5: 1 to 5.

8. The process for the preparation of partially anticoagulant heparin derivatives according to claim 4, wherein: before step 3), the pH is first adjusted to 7.0.

9. The process for the preparation of partially anticoagulant heparin derivatives according to claim 8, wherein: in the step 3), the membrane filtration is any one of dialysis, ultrafiltration and tangential flow filtration, and when ultrafiltration is selected, the pore size of the membrane is 0.5 kDa-2 kDa.

10. Use of a partially anticoagulant heparin derivative according to any one of claims 1 to 3 for the preparation of a medicament with anticoagulant and/or anti-inflammatory activity.