Application of unsaturated hyaluronic acid tetrasaccharide in preparation of product for increasing platelet quantity or platelet hematocrit

Abstract

The invention relates to the technical field of medicines, in particular to application of unsaturated hyaluronic acid tetrasaccharide to preparation of a product for increasing the number of platelets or the hematocele of the platelets. Animal experiments show that the unsaturated hyaluronan tetrasaccharide can significantly increase the number of platelets in mouse blood and platelet hematocrit for the first time. In addition, the platelet distribution width and the large platelet ratio can be reduced. A further research shows that the unsaturated hyaluronic acid tetrasaccharide has good tolerance and has high selectivity on the action of platelets in blood. The unsaturated hyaluronic acid tetrasaccharide disclosed by the invention has an important application prospect in preparation of a product for increasing the number of platelets or the hematocele of the platelets.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of unsaturated hyaluronic acid tetrasaccharide in the preparation of products for increasing platelet count or platelet volume. Background Art

[0002] Platelets are important members of the blood and play a key role in hemostasis and coagulation (DOI: 10.1038 / s41569-018-0110-0). Studies have shown that cirrhosis, blood tumors, chemotherapy, infection, drug treatment, etc. can cause thrombocytopenia (DOI: 10.3760 / cma.j.issn.1671-0282.2022.02.005). Thrombocytopenia can lead to adverse thrombosis and bleeding reactions, and even be life-threatening in severe cases (DOI: 10.1038 / s41569-018-0110-0). Thrombocytopenia is very common in clinical emergencies, with an incidence of about 10%; among them, the incidence in elderly patients is even higher, about 23% (DOI: 10.3760 / cma.j.issn.1671-0282.2022.02.005). At present, the drugs and biological agents that increase platelets clinically mainly include recombinant human thrombopoietin, recombinant human interleukin-11, thrombopoietin receptor agonists, etc. (DOI: 10.1038 / s41569-018-0110-0). However, the above products have many limitations in clinical application (DOI: 10.1038 / s41569-018-0110-0). For example, they must be injected and used in hospitals, etc. In addition, some patients also have other side effects such as allergies and poor tolerance (DOI: 10.1007 / s12185-018-2445-z; DOI:10.7150 / jca.26690). Therefore, it is of great significance to develop products that can increase platelets that are safe, effective, easy to use, and have few side effects.

[0003] Hyaluronic acid, also known as hyaluronic acid, is a glycosaminoglycan composed of alternating repeating units of D-glucuronic acid and N-acetylamino-D-glucose disaccharide (DOI: 10.3390 / biom10111525). Hyaluronic acid is widely used in medicine, food, and health care (DOI: 10.1016 / j.carbpol.2021.118320). Studies have shown that hyaluronic acid tetrasaccharide can promote the body's immune response (DOI:10.1016 / j.carbpol.2021.118699). In addition, it can also induce the differentiation of human epidermal keratinocytes (DOI: 10.1111 / ics.12105). However, whether hyaluronic acid tetrasaccharide can increase platelet count and platelet volume has not been studied.

[0004] This study relied on the patented method previously developed by the inventor (China invention patent, ZL 2023 1 0238846.8) to prepare unsaturated hyaluronic acid tetrasaccharide. Through in vivo animal experiments, we found for the first time that unsaturated hyaluronic acid tetrasaccharide can significantly increase platelet count and platelet volume, and has broad application development prospects in the prevention and treatment of thrombocytopenia. Summary of the invention

[0005] Based on the deficiencies in the prior art, the present invention discloses an application of unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing platelet count or platelet volume.

[0006] The present invention adopts the following technical solution: The present invention provides for the first time an application of unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing platelet count or platelet hematocrit.

[0007] Furthermore, the unsaturated carbon-carbon double bonds of the unsaturated hyaluronic acid tetrasaccharide are located at the C4 and C5 positions of the non-reducing end glucuronic acid fragment, and its chemical structure is:

[0008] Where R is H / Na / K.

[0009] Furthermore, the use of the unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing platelet count or platelet hematocrit is characterized in that the unsaturated hyaluronic acid tetrasaccharide can increase platelet count or platelet hematocrit, The unsaturated hyaluronic acid tetrasaccharide can increase the number of platelets in the blood, and the dosage is 200 mg / kg / day to 400 mg / kg / day; The unsaturated hyaluronic acid tetrasaccharide can increase platelet volume, and the dosage is 200 mg / kg / day to 400 mg / kg / day; Furthermore, the use of the unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing platelet count or platelet volume is characterized in that the unsaturated hyaluronic acid tetrasaccharide can reduce the platelet distribution width or the average large platelet ratio, The unsaturated hyaluronic acid tetrasaccharide can significantly reduce platelet distribution width, with a dosage of 100 mg / kg / day; The unsaturated hyaluronic acid tetrasaccharide can significantly reduce the ratio of large platelets, with a dosage of 200 mg / kg / day; Furthermore, the unsaturated hyaluronic acid tetrasaccharide has good tolerance and high safety, and its effect on platelets in the blood is highly selective.

[0010] Another aspect of the present invention provides a use of unsaturated hyaluronic acid tetrasaccharide in the preparation of a drug for preventing or treating thrombocytopenia.

[0011] Furthermore, the disease is insufficient platelet production, excessive platelet destruction, abnormal platelet distribution, preferably, aplastic anemia (AA), acute leukemia (AL), infection, primary immune thrombocytopenia (ITP), disseminated intravascular coagulation (DIC), thrombotic thrombocytopenia (TTP), heparin-induced thrombocytopenia (HIT), systemic lupus erythematosus (SLE), hypersplenism or splenomegaly causing a decrease in platelet count.

[0012] Furthermore, the unsaturated carbon-carbon double bonds of the unsaturated hyaluronic acid tetrasaccharide are located at the C4 and C5 positions of the non-reducing end glucuronic acid fragment, and its structural formula is:

[0013] Where R is H / Na / K.

[0014] The third aspect of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains a therapeutic dose of unsaturated hyaluronic acid tetrasaccharide and a pharmaceutically acceptable carrier; the preferred pharmaceutical composition is tablets, oral liquids, aerosols, pills, capsules, granules, ointments, pellets, syrups, powders, granules, tinctures, powder injections or injections.

[0015] Compared with the prior art, the present invention has the following positive effects: The present invention discloses an application of unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing the number of platelets or the hematocrit. The unsaturated hyaluronic acid tetrasaccharide is convenient for oral administration, has good safety, and is highly tolerable, can significantly increase the number of platelets or the hematocrit in the blood, and has broad application development prospects in the prevention or treatment of thrombocytopenia and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The following are the statistical graphs of the weight changes of mice during the experiment and the length of the colorectum of mice after the experiment: A is the weight change graph; B is the statistical graph of the length of the colorectum after 4 weeks of administration. One-way ANOVA was used for statistical analysis between the groups, and the statistical results showed that there was no significant difference between the groups (P > 0.05).

[0017] Figure 2 The following are the statistical diagrams of immune-related cells in the blood of mice after 4 weeks of administration: A is the statistical diagram of white blood cells (WBC); B is the statistical diagram of neutrophils (NEUT); C is the statistical diagram of lymphocytes (LYM); D is the statistical diagram of monocytes (MONO); E is the statistical diagram of eosinophils (EO); and F is the statistical diagram of basophils (BASO). One-way ANOVA was used for statistical analysis between the groups, and the statistical results showed that there was no significant difference between the groups (P > 0.05).

[0018] Figure 3 The following are the statistical graphs of red blood cell-related indexes in the blood of mice after 4 weeks of administration: A is the statistical graph of the number of red blood cells (RBC); B is the statistical graph of hemoglobin (HGB); C is the statistical graph of hematocrit (HCT); D is the statistical graph of mean corpuscular volume (MCV); E is the statistical graph of mean corpuscular hemoglobin (MCH); F is the statistical graph of mean corpuscular hemoglobin concentration (MCHC); G is the statistical graph of red blood cell distribution width SD (RDW-SD); H is the statistical graph of reticulocyte (RET) number; I is the statistical graph of immature reticulocyte index (IRF). One-way ANOVA was used for statistical analysis between the groups, and the statistical results showed that there was no significant difference between the groups (P > 0.05).

[0019] Figure 4The following are the statistical graphs of platelet-related indexes in the blood of mice after 4 weeks of administration: A is the statistical graph of platelet (PLT) number; B is the statistical graph of platelet hematocrit (PCT); C is the statistical graph of platelet distribution width (PDW); D is the statistical graph of large platelet ratio (P-LCR); E is the statistical graph of mean platelet volume (MPV). One-way ANOVA was used for statistical analysis among the groups, where * is P < 0.05, ** is P < 0.01, *** is P < 0.001, and **** is P < 0.0001. DETAILED DESCRIPTION

[0020] Embodiments of the present invention are described in detail below in conjunction with specific examples. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] Example 1: Preparation of unsaturated hyaluronic acid tetrasaccharide (1) Preparation of culture medium According to the patent method previously developed by the inventor (China invention patent, ZL 2023 1 0238846.8) and the publicly published paper (DOI: 10.1016 / j.carbpol.2024.122074), unsaturated hyaluronic acid tetrasaccharide was prepared by fermentation degradation using Bacteroides salierii strain CSP6. The components of the culture medium of Bacteroides salierii strain CSP6 are as follows: Hyaluronic acid 2 g / L, tryptone 3 g / L, peptone 3 g / L, yeast extract 3 g / L, mucin 0.5 g / L, bile salt No. 3 0.4 g / L, cysteine ​​hydrochloride 0.8 g / L, heme 0.05 g / L, Tween 80 1 mL / L, sodium chloride 4.5 g / L, potassium chloride 2.5 g / L, magnesium chloride 4.5 g / L, calcium chloride 0.2 g / L, potassium dihydrogen phosphate 0.4 g / L, trace elements 2 mL / L. The final concentration of the trace elements is as follows: 3.0 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium chloride dihydrate, 0.32 g / L manganese chloride tetrahydrate, 0.1 g / L ferrous sulfate heptahydrate, 0.18 g / L cobalt sulfate heptahydrate, 0.18 g / L zinc sulfate heptahydrate, 0.01 g / L copper sulfate pentahydrate, 0.092 g / L nickel chloride hexahydrate. The medium is prepared with distilled water, and the pH value is 6.4-6.5.

[0022] (2) Inoculation, culture and fermentation preparation After the medium is prepared, it is poured into an anaerobic vial and sterilized with nitrogen. First, the Bacteroides salierii strain CSP6 is inoculated into the above medium and cultured anaerobically (80% N2, 10% H2 and 10% CO2) at 25-42 °C for 1-4 days to obtain a seed solution. Further, the seed solution is inoculated into the culture medium at an inoculum volume ratio of 1% to 10%, and cultured anaerobically (80% N2, 10% H2 and 10% CO2) at 25-42 °C for 1-4 days. During the culture process, the substrate utilization and product expression are tracked and detected by thin layer chromatography (TLC); (3) Isolation and purification of unsaturated hyaluronic acid tetrasaccharide The fermentation liquid was separated into solid and liquid and the supernatant was taken, concentrated, sterilized, filtered and freeze-dried. Ultrapure water was used as the mobile phase, and Bio-Gel P-2 Gel gel column was used for separation and purification, and freeze-dried to obtain unsaturated hyaluronic acid tetrasaccharide. The structure was verified by mass spectrometry and nuclear magnetic resonance analysis, and then used in animal experiments.

[0023] Example 2: Application of unsaturated hyaluronic acid tetrasaccharide in the preparation of products for increasing platelet count or platelet hematocrit (1) Animal experiments Male SPF C57BL / 6J mice (7 weeks old) were purchased. The animal breeding temperature was maintained at 22-25 ℃ and the light was maintained for 12 hours per day. The mice were given free water and food and fed adaptively for 7 days. The weight of the mice was observed and recorded every day. The experiment began when the mice were 8 weeks old. The mice were randomly divided into 4 experimental groups according to the standard of 7 mice per group (n=7): 1) Normal control group (NC group, without unsaturated hyaluronic acid tetrasaccharide); 2) unsaturated hyaluronic acid tetrasaccharide low-dose group (HA4-L group, oral dose of 100 mg / kg / day); 3) medium-dose group of unsaturated hyaluronic acid tetrasaccharide (HA4-M group, oral dose of 200 mg / kg / day); 4) High-dose group of unsaturated hyaluronic acid tetrasaccharide (HA4-H group, oral dose of 400 mg / kg / day).

[0024] Each group of mice was given sterile mouse maintenance feed and free access to sterile water. On day 0, gavage was started with a gavage volume of 0.10 mL, and the normal control group was gavaged with 0.10 mL of sterile saline. Body weight was recorded every day during the experiment, and the weight of the mice on day 0 was taken as the initial body weight. After 4 weeks of administration, the experiment ended. At the end of the experiment, blood and colorectal samples of the mice were collected for subsequent analysis.

[0025] (2) Platelet level analysis An automatic blood analyzer was used to detect various blood indicators of mice after four weeks of administration, including white blood cell count (WBC), neutrophil count (NEUT), lymphocyte count (LYM), monocyte count (MONO), eosinophil count (EO), basophil count (BASO), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width SD (RDW-SD), reticulocyte count (RET), immature reticulocyte index (IRF), platelet count (PLT), platelet hematocrit (PCT), platelet distribution width (PDW), large platelet ratio (P-LCR) and mean platelet volume (MPV).

[0026] (3) Experimental results and analysis All experimental data were plotted by GraphPad Prism, and the results were expressed as Mean±SEM. One-way ANOVA was used for statistical analysis among the groups, where * means P < 0.05, ** means P < 0.01, *** means P < 0.001, and **** means P < 0.0001.

[0027] like Figure 1 As shown in A, after continuous administration of unsaturated hyaluronic acid tetrasaccharide for 4 weeks, there was no significant difference in the body weight of mice in each group.

[0028] like Figure 1 As shown in B, after continuous administration of unsaturated hyaluronic acid tetrasaccharide for 4 weeks, there was no significant difference in the colorectal length of mice in each group.

[0029] Figure 1 The results of the study showed that unsaturated hyaluronic acid tetrasaccharide is well tolerated and highly safe. Oral administration of unsaturated hyaluronic acid tetrasaccharide does not affect the normal growth of mice and does not produce intestinal toxicity.

[0030] like Figure 2 As shown, after continuous administration of unsaturated hyaluronan tetrasaccharide for 4 weeks, there was no significant difference in the white blood cell count (WBC), neutrophil count (NEUT), lymphocyte count (LYM), monocyte count (MONO), eosinophil count (EO), and basophil count (BASO) of mice in each group.

[0031] The above research results further show that unsaturated hyaluronic acid tetrasaccharide has good tolerance and high safety, and will not stimulate the body to produce immune inflammatory response at the administered dose.

[0032] like Figure 3As shown, after continuous administration of unsaturated hyaluronan tetrasaccharide for 4 weeks, there were no significant differences in red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width SD (RDW-SD), reticulocyte count (RET) and immature reticulocyte index (IRF) among the mice groups.

[0033] The above research results further show that unsaturated hyaluronic acid tetrasaccharide is well tolerated and highly safe. It does not affect the number of red blood cells and hemoglobin content in the blood of mice at the administered dose.

[0034] like Figure 4 As shown in A, compared with the mice in the NC group, the platelet count (PLT) of the mice in the drug-treated group increased significantly in a dose-dependent manner, indicating that unsaturated hyaluronic acid tetrasaccharide has the effect of increasing platelet levels.

[0035] like Figure 4 As shown in B, compared with the mice in the NC group, the platelet hematocrit (PCT) of the mice in the HA4-M group and the HA4-H group increased significantly, indicating that unsaturated hyaluronic acid tetrasaccharide has the effect of increasing platelet hematocrit.

[0036] like Figure 4 As shown in C, the platelet distribution width (PDW) of the HA4-L group mice was significantly reduced compared with that of the NC group mice, indicating that the administration of unsaturated hyaluronan tetrasaccharide increased the homogeneity of platelets.

[0037] like Figure 4 As shown in D, the large platelet ratio (P-LCR) of mice in the HA4-M group was significantly reduced compared with that in the NC group. During the development of platelets, their volume gradually decreases and eventually matures. The above research results suggest that unsaturated hyaluronan tetrasaccharide reduces platelet volume and promotes platelet maturation.

[0038] like Figure 4 As shown in E, there was no significant difference in mean platelet volume (MPV) among the mice groups.

[0039] In summary, the unsaturated hyaluronic acid tetrasaccharide provided by the present invention is convenient for oral administration, has good safety and high tolerance. Studies have shown that it can significantly increase the number of platelets or platelet volume in the blood. The unsaturated hyaluronic acid tetrasaccharide will not stimulate the body to produce an immune inflammatory response, will not affect the number of white blood cells, red blood cells and lymphocytes in the blood of mice, has a high degree of selectivity for the effect on platelets, and has broad application development prospects in the prevention or treatment of related diseases such as thrombocytopenia.

[0040] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Application of an unsaturated hyaluronic acid tetrasaccharide in the preparation of a product for increasing platelet count or platelet hematocrit.

2. The use of an unsaturated hyaluronic acid tetrasaccharide according to claim 1 in the preparation of a product for increasing platelet count or platelet hematocrit, characterized in that: The unsaturated carbon-carbon double bonds of the unsaturated hyaluronic acid tetrasaccharide are located at the C4 and C5 positions of the non-reducing end glucuronic acid fragment, and its structural formula is: , Where R is H / Na / K.

3. The use of an unsaturated hyaluronic acid tetrasaccharide according to claim 1 in the preparation of a product for increasing platelet count or platelet hematocrit, characterized in that: The unsaturated hyaluronic acid tetrasaccharide can increase the platelet count or platelet volume.

4. The use of an unsaturated hyaluronic acid tetrasaccharide according to claim 1 in preparing a product for increasing platelet count or platelet hematocrit, characterized in that: The unsaturated hyaluronan tetrasaccharide can reduce the platelet distribution width or the average large platelet ratio.

5. Use of the unsaturated hyaluronic acid tetrasaccharide according to claim 1 in the preparation of a drug for preventing or treating thrombocytopenia.

6. The use according to claim 5, characterized in that The diseases are insufficient platelet production, excessive platelet destruction, and abnormal platelet distribution, preferably, aplastic anemia (AA), acute leukemia (AL), and infection, primary immune thrombocytopenia (ITP), disseminated intravascular coagulation (DIC), thrombotic thrombocytopenia (TTP), heparin-induced thrombocytopenia (HIT), systemic lupus erythematosus (SLE), hypersplenism or splenomegaly causing a decrease in platelet count.

7. The use according to claim 5, characterized in that The unsaturated carbon-carbon double bonds of the unsaturated hyaluronic acid tetrasaccharide are located at the C4 and C5 positions of the non-reducing end glucuronic acid fragment, and its structural formula is: ， Where R is H / Na / K.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutic dose of unsaturated hyaluronic acid tetrasaccharide and a pharmaceutically acceptable carrier; the preferred pharmaceutical composition is tablets, oral liquids, aerosols, pills, capsules, granules, pastes, pellets, syrups, powders, granules, tinctures, powder injections or injections.

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