Hyaluronic acid derivatives, methods for producing the same, and use

Abstract

The present invention relates to a hyaluronic acid derivative represented by formula I, as well as a method for producing the same and its use in the production of an anti-infective agent. The compound of the present invention or a pharmaceutically acceptable salt thereof differs significantly in structure from commercially available hyaluronic acid substances, such as TCIsHA and R&D HA, and can form a molecular structure identified by HepSS-1. This novel molecule can exert an anti-infective effect by inhibiting the binding of the SARS-CoV-2 spike protein to ACE2 in host cells, and is less likely to cause serious adverse events such as bleeding and thrombocytopenia in clinical practice compared to heparin. [Formula 1] JPEG2026521621000007.jpg70170

Description

Technical Field

[0001] This application claims the priority of a prior application filed with the China National Intellectual Property Administration on June 29, 2023, with a patent application number of 202310778756.8 and an invention title of "Hyaluronic Acid Derivatives, Their Manufacturing Methods and Uses". The entire content of the above application is incorporated herein by reference.

[0002] The present invention belongs to the field of medicinal chemistry, and specifically relates to hyaluronic acid derivatives, their manufacturing methods and uses.

Background Art

[0003] Hyaluronic acid (HA) is a glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine with alternating β-1,4-glycosidic bonds and β-1,3-glycosidic bonds. It is an important component in the natural extracellular matrix. Since hyaluronic acid substances vary depending on the molecular weight and can bring different pharmacological activities depending on the modifying groups, they can be widely used in the fields of cosmetics, food, and pharmaceuticals.

[0004] Due to its high water absorption capacity and high viscoelasticity, hyaluronic acid has unique properties in biomaterials and is suitable for various medical and pharmaceutical uses. Various hyaluronic acid products can be found in daily life. For example, since hyaluronic acid can retain moisture, it is used in some cosmetics to maintain the youthfulness and freshness of the skin. Although the content of hyaluronic acid in the skin is abundant, with aging, the water retention ability of hyaluronic acid in the skin decreases due to depolymerization. As can be seen from the above, maintaining abundant hyaluronic acid in the skin can contribute to maintaining youthfulness.

[0005] In fields other than cosmetics, researchers have proposed many ideas for using hyaluronic acid, but its actual use in medicine is not extensive, mainly for the treatment of osteoarthritis and use in ophthalmic medical devices.

[0006] Heparin is a type of glycosaminoglycan, in which hexuronic acid and N-acetylglucosamine are alternately formed by 1,4-glycosidic bonds. It is already widely used clinically as an anticoagulant, and several modified hyaluronic acid compounds have anticoagulant effects similar to heparin. However, the anticoagulant activity of heparin and its analogs can easily cause serious adverse events such as bleeding and thrombocytopenia. [Overview of the Initiative]

[0007] In order to improve upon the technical problems of the prior art, in a first aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, the structure of which is shown below.

[0008] [ka] In the above formula I, R is homologous or different, and is independently selected from H or SO3H. M is selected from a proton or a monovalent cation. n is chosen from 3 to 9.

[0009] According to embodiments of the present invention, n is selected from 3, 4, 5, 6, 7, 8, and 9.

[0010] According to embodiments of the present invention, the monovalent cation is Na + or K + They are selected from among them.

[0011] According to embodiments of the present invention, the weight-average molecular weight of formula I is less than 10,000. In some embodiments, the weight-average molecular weight of formula I is between 2,480 and 6,590.

[0012] According to embodiments of the present invention, in formula I, at least some of the R are selected from SO3H (i.e., not all R are H). In some embodiments, in formula I, the degree of sulfation substitution is 4.2 or less; in some embodiments, in formula I, the degree of sulfation substitution is greater than 3.65, preferably 3.75 or more; in some embodiments, in formula I, the degree of sulfation substitution is 4.2 or less and greater than 3.65; in some embodiments, in formula I, the degree of sulfation substitution is 4.2 or less and 3.75 or more; in some embodiments, in formula I, the degree of sulfation substitution is 4.0 or less and 3.75 or more; for example, the degree of sulfation substitution is selected from 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, 4.00, 4.05, 4.10, 4.05, 4.10, 4.15 or 4.20.

[0013] According to embodiments of the present invention, in formula I above, NHCOCH3 is not sulfurized.

[0014] According to embodiments of the present invention, the structure of formula I can be identified by HepSS-1.

[0015] In a second aspect, the present invention relates to a method for producing the compound of formula I or a pharmaceutically acceptable salt thereof, (1) A step of mixing water with hyaluronic acid or a water-soluble salt thereof, (2) The step of adding a sulfating agent and reacting, (3) Add ethanol, mix, and centrifuge. (4) Remove the supernatant, add water, add ethanol, mix, and centrifuge. (5) Remove the supernatant and leave it for 4 to 24 hours, (6) A method is provided which includes the steps of adding water, filtering, freeze-drying, and obtaining the product.

[0016] According to an embodiment of the present invention, the sulfating agent is selected from complexes of sulfur trioxide, and preferably, it is a pyridine complex of sulfur trioxide.

[0017] According to an embodiment of the present invention, the hyaluronic acid or its water-soluble salt is selected from Hyalonano, and preferably, the hyaluronic acid or its water-soluble salt is purchased from Kewpie.

[0018] According to an embodiment of the present invention, in the above step (1), it is mixed with 0.5 to 3 g of hyaluronic acid or its water-soluble salt per 1 mL of water, preferably, it is mixed with 0.8 to 1.2 g of hyaluronic acid or its water-soluble salt per 1 mL of water. For example, it is mixed with 1 g of hyaluronic acid or its water-soluble salt per 1 mL of water.

[0019] According to an embodiment of the present invention, the water-soluble salt is selected from sodium hyaluronate.

[0020] According to an embodiment of the present invention, in the above step (2), the reaction temperature is 0 to 20 °C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 °C, preferably, it is 3 to 10 °C, the reaction time is 24 to 100 hours, preferably, it is 36 to 90 hours, for example, 36, 48, 60, 72, 84 hours.

[0021] According to an embodiment of the present invention, the mass ratio of the sulfating agent and hyaluronic acid or its water-soluble salt is 0.5 to 6:100, preferably, it is 1 to 3:100, for example, 1.5:100, 2.0:100, 2.5:100, 3.0:100, 3.5:100, 4.0:100.

[0022] According to an embodiment of the present invention, in the above step (3), regarding the addition amount of ethanol, 10 to 50 mL of ethanol is added per 1 g of hyaluronic acid or its water-soluble salt, preferably, 20 to 30 mL of ethanol is added, for example, 25 mL of ethanol is added.

[0023] According to an embodiment of the present invention, in the above step (4), regarding the amount of water added, 0.5 to 2 mL of water is added per 1 g of hyaluronic acid or its water-soluble salt, preferably 1 mL of water is added, and regarding the amount of ethanol added, 20 to 30 mL of ethanol is added per 1 g of hyaluronic acid or its water-soluble salt, preferably 25 mL of ethanol is added.

[0024] According to an embodiment of the present invention, in the above steps (3) and (4), centrifugation is performed at 0 to 10°C and 2000 to 4000 rpm for 5 to 30 minutes. Preferably, the centrifugation temperature is 3 to 6°C, for example, 4°C, preferably the centrifugation rotation speed is 3000 rpm, and preferably the centrifugation time is 10 to 20 minutes, for example, 15 minutes.

[0025] According to an embodiment of the present invention, in the above step (5), the standing temperature is -20 to 5°C, preferably -10 to 4°C, and the standing time is preferably 6 to 12 hours.

[0026] According to an embodiment of the present invention, in the above step (6), regarding the amount of water added, 0.5 to 2 mL of water is added per 1 g of hyaluronic acid or its water-soluble salt, preferably 1 mL of water is added.

[0027] According to an embodiment of the present invention, in the above step (6), a disk filter is employed, preferably a 0.45 μm disk filter is used.

[0028] In a third aspect, the present invention further provides a composition containing the compound of the above formula I or its pharmaceutically acceptable salt.

[0029] According to an embodiment of the present invention, the above composition further contains a pharmaceutically acceptable carrier.

[0030] In a fourth aspect, the present invention provides the use of the compound of formula I or a pharmaceutically acceptable salt thereof or the composition thereof in the manufacture of an anti-infective agent.

[0031] According to embodiments of the present invention, the above infection is selected from infections caused by coronavirus (COVID-19), and the above coronavirus is selected from SARS-CoV-2.

[0032] According to embodiments of the present invention, the drug formulation may be administered topically to the skin, selected from, for example, creams, patches, ointments, cream formulations, gels, and sprays, or it may be administered orally (i.e., as an oral formulation), in a solid form selected from, for example, tablets, granules, capsules, pills, dripping pills, etc. In some embodiments, the drug formulation may also be selected from liquid or semi-solid dosage forms (liquids, suspensions, solutions, dispersions, ointments, creams, emulsions, microemulsions, sprays, patches, and infusions). Injectable, parenteral, inhalation, oral, and nasal formulations are also within the scope of the present invention.

[0033] The above formulation further comprises a physiologically acceptable carrier (e.g., a biocompatible material), and optionally includes, for example, a surfactant, an excipient, a humectant, an emulsifier, a suspending agent, a salt or buffer for adjusting osmotic pressure, a colorant, an essence, a stabilizer, a bactericide, a preservative, or other common supplements.

[0034] In a fifth aspect, the present invention provides an anti-infective method comprising administering to a patient a therapeutically effective amount of the compound of formula I of the present invention or a pharmaceutically acceptable salt thereof or the composition thereof.

[0035] According to embodiments of the present invention, the above infection is selected from infections caused by coronavirus (COVID-19). Beneficial Effects of the Present Invention

[0036] This invention has found that the compound of formula I or a pharmaceutically acceptable salt thereof differs significantly in structure from commercially available hyaluronic acid substances, such as TCIsHA and R&D HA, and can form a molecular structure identified by HepSS-1. This novel molecule can exert an anti-infective effect by inhibiting the binding of the SARS-CoV-2 spike protein to ACE2 in host cells, and is less likely to cause serious adverse events such as bleeding and thrombocytopenia in clinical practice compared to heparin. [Brief explanation of the drawing]

[0037] [Figure 1] The 1H NMR nuclear magnetic spectrum of sHA is shown. [Figure 2] The results of the HepSS-1 discrimination experiment are shown. [Figure 3] The HPLC pattern of sHA synthesized by the present invention is shown, where the numbers represent the number of sugar residues, and the arrows indicate the same number of disaccharide units (the numbers 2, 4, 6, etc. represent the number of sugar residues, the solid arrows indicate screening up to higher MV with higher sulfate, and the dotted arrows indicate that the maximum MW of sHA is approximately the same as that of Fragmin). [Figure 4] The results of binding experiments between the compound of the present invention and the SARS-CoV-2 spike protein are shown. [Figure 5] The prothrombin times for sHA and the control group substance are shown. [Modes for carrying out the invention]

[0038] The technical aspects of the present invention will be described in more detail below, in accordance with specific embodiments. The embodiments described below are merely illustrative and interpretable to illustrate the present invention, and should not be interpreted as limiting the scope of the claims. Any technology realized based on the above-described aspects of the present invention falls within the scope of the claims according to the present invention.

[0039] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or may be manufactured by known methods.

[0040] Example 1 Manufacturing of the compound of the present invention 1.1 Method for producing compound sHA of formula I 1.11 Preparation of compound sHA-1 of formula I 20 mL of deionized water was mixed with 20 g of hyaluronic acid (trade name Hyalonano, Kewpie 38399), then 0.3 g of pyridine-sulfur trioxide complex (purchased from FUJIFILM Wako Pure Chemical Corporation) and mixed at 4°C for 72 hours. 500 mL of ethanol was added and mixed, then centrifuged (4°C, 3000 rpm, 15 minutes). The supernatant was removed, 20 mL of deionized water was added, 500 mL of ethanol was added and mixed, then centrifuged (4°C, 3000 rpm, 15 minutes). The supernatant was removed, 20 mL of deionized water was added, 500 mL of ethanol was added and mixed, then centrifuged (4°C, 3000 rpm, 15 minutes). The supernatant was removed and the mixture was stored in the refrigerator overnight. 20 mL of deionized water was added, and the solution was applied to a disc filter (0.45 μm: ADVANTEC). The solution was then freeze-dried to obtain 1.73 g of product (i.e., sHA-1).

[0041] 1.12 Referring to the process in 1.11, different pyridine-sulfur trioxide complexes were selected in mass ratios with hyaluronic acid to prepare compounds sHA-2 to sHA-7 of formula I.

[0042] 1.2 Structural identification of compound sHA of formula I 1.2.1 Measurement Method (1) 1 1H NMR (2)HPLC (3) HepSS-1 identification (based on ELISA) The samples used are shown in the table below.

[0043] [Table 1]

[0044] Of these, highly sulfated hyaluronic acid (TCIs HA) can also be manufactured by referring to the literature [Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions. Taichi Miuraa, Noriyuki Yuasac, Hayato Otab, Masato Habuc, Mitsuko Kawanoa, Fumiaki Nakayamaa, Shoko Nishihara. August 2019, Biochemical and Biophysical Research Communications 518(3)].

[0045] The steps of the method are as follows:

[0046] Each sample (20 μg / 100 μL) was immobilized in a 96-well plate. Incubate with HepSS-1, Incubate with HRP-labeled anti-mouse IgM. Incubate with 3,3'-diaminobenzidine (DAB) and 30% hydrogen peroxide. iMark plate reader wavelength: 405nm.

[0047] 1.2.2 Analysis of Identification Results Upon identification, the structures of compounds sHA-1 to sHA-7 are shown below.

[0048] [ka] In the above structure, R is either H or SO3H, GlcA is glucuronic acid, GlcNac is N-acetylglucosamine, and n=3.

[0049] [Table 2]

[0050] (1) 1 Identified by 1H NMR and HPLC, in the structures of sHA-1 to sHA-7 described above, n is 3 (8 mers), the weight-average molecular weight (MW) is 2480, and the degree of sulfate substitution (i.e., the [SO3] of each disaccharide) is - The number of groups was 4.2 or less and greater than 3.65 (the degree of sulfate substitution for disaccharides with both ends completely sulfated is 5). In TCIsHA, the degree of sulfate substitution was 3.65.

[0051] (2) 1 ¹H NMR showed that in the structures of sHA-1 to sHA-7 described above, the NHCOCH3 of GlcNac was not substituted with SO3-, yet these sHAs could be identified by HepSS-1 (Figure 2). HepSS-1 identified the N-sulfation of heparan sulfate but not the O-sulfation of heparan sulfate, heparin, or hyaluronic acid. Some O-SO3- groups tightly bound three-dimensionally to NHCOCH3 in GlcNac, forming molecular structures that could be identified by HepSS-1. The HepSS-1 identification experiment results further showed that sHAs could be identified by HepSS-1, but neither TCIsHA nor R&DHA could be identified by HepSS-1 (see Figures 1-2).

[0052] 1.3 Preparation and identification of other compounds Experiments have verified that products with a similar degree of sulfate substitution can be produced using hyaluronic acid with other molecular weights, for example, hyaluronic acid with n=3, where n is 3-9 (8-20 mers) and weight-average molecular weight (MW) is 2480-6590, by referring to the process in 1.11 (see Figure 3). 1The 1H NMR spectrum shows that any disaccharide can be identified by HepSS-1 if n is 3-9 (8-20 mers), weight-average molecular weight (MW) is 2480-6590, and the degree of sulfate substitution (i.e., the number of [SO3-] groups in each disaccharide) is <4.2 and >3.65 (the degree of sulfate substitution for disaccharides with both ends completely sulfated is 5).

[0053] Example 2 Physiological activity of the compound of the present invention 2.1 Anti-SARS-CoV-2 infection test The binding activity of the sHA of the present invention to the spike protein of SARS-CoV-2 was evaluated by immunoassay based on ELISA, and the test steps were as follows.

[0054] sHA-1 (20 μg / 100 μL, 20 μg / 100 μL) prepared according to Example 1 was added to a SARS-CoV-2 spike protein coated 96-well plate. The antibody was incubated with HepSS-1 (anti-N-heparan sulfate, mouse IgM), and two doses of anti-immunoglobulin were added. Incubate with 3,3'-diaminobenzidine (DAB), iMark plate reader wavelength: 405nm.

[0055] As can be seen from the experimental results (see Figure 4), the compound of the present invention can exert an anti-infective effect by inhibiting the binding of the SARS-CoV-2 spike protein to ACE2 on host cells.

[0056] 2.2 Effects of the Compounds of the Present Invention on Blood Coagulation Based on the reference [Sulakshan Sulakshana et al., Anesth Essays Res., 2021, 15(4):341-347], the anticoagulant activity of the compound was evaluated by measuring prothrombin time.

[0057] The experimental method and steps are as follows:

[0058] 1) Approximately 2 mL of blood was obtained from the abdominal aorta of male SD rats (8 weeks old). One sample per rat, n=3.

[0059] 2) Each substance was added to blood and mixed at a concentration of 27.8 μg / mL or 278 μg / mL (27.8 μg / mL is typically used for blood transfusions).

[0060] 3) To evaluate the anticoagulant effect of the samples, prothrombin time was measured using CoaguChek X (Sekisui Medical).

[0061] Control group: 1) Physiological saline solution, 2) Unfractionated heparin (UFH), heparin sodium injection solution 5000 units / 5 mL, Mochida (average molecular weight 15000), 3) Fragmin: Low molecular weight heparin, average molecular weight 5000, 5000 units / 5mL, 4) Arixtra: Low molecular weight heparin, average molecular weight 1728 (5 mers), 5000 units / 5 mL, 5) No additives.

[0062] The test results are shown in Figure 5. At 27.8 μg / mL, there was no change due to the substance. At 278 μg / mL, the prothrombin time for UFH, Fragmin, or Arixtra was significantly longer than in the saline group and the control group. However, sHA-1 manufactured based on Example 1 was unaffected. Of these, Fragmin and Arixtra are heparin drugs with low molecular weight, and their purpose is to reduce anticoagulant activity. However, according to existing studies, they bind relatively weakly to the SARS-CoV-2 spike protein. Even at 10 times the clinically used concentration of heparin, sHA-1 did not show any anticoagulant activity. Therefore, compared to heparin and, more specifically, low molecular weight heparin, the sHA of the present invention is less likely to cause serious adverse events such as bleeding and thrombocytopenia in clinical practice.

[0063] Embodiments of the present invention have been described above. However, the present invention is not limited to the embodiments described above. Any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, wherein the structure of formula I is shown below, 【Chemistry 1】 In the above formula I, R is homologous or different, and is H or SO independently of each other. ３ Selected from H, M is selected from a proton or a monovalent cation. n is selected from 3 to 9. In the above formula I, the degree of sulfation substitution is 4.2 or less and greater than 3.

65. The structure of formula I can be identified by HepSS-1. A compound of formula I or a pharmaceutically acceptable salt thereof.

2. In the above formula I, NHCOCH ３ It is not sulfated or substituted, Preferably, n is selected from 3, 4, 5, 6, 7, 8, 9. Preferably, the monovalent cation is Na ＋ or K ＋ Selected from, A compound of formula I as described in claim 1, or a pharmaceutically acceptable salt thereof.

3. In the above formula I, the degree of sulfation substitution is 4.2 or less and 3.75 or more. A compound of formula I according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. A method for producing a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, (1) A step of mixing water with hyaluronic acid or a water-soluble salt thereof, (2) The step of adding a sulfating agent and reacting, (3) Add ethanol, mix, and centrifuge, (4) Remove the supernatant, add water, add ethanol, mix, and centrifuge. (5) Remove the supernatant and leave it for 4 to 24 hours, (6) The steps include adding water, filtering, freeze-drying, and obtaining the product, method.

5. The sulfurizing agent is characterized by being selected from pyridine complexes of sulfur trioxide. The method according to claim 4.

6. In step (1) above, the mixture is characterized by mixing 0.5 to 3 g of hyaluronic acid or a water-soluble salt thereof with 1 mL of water. The method according to claim 4.

7. In step (2) above, the reaction temperature is 0 to 20°C, and the reaction time is 24 to 100 hours. The mass ratio of the sulfating agent to hyaluronic acid or its water-soluble salt is 0.5 to 6:

100. In step (3) above, regarding the amount of ethanol to be added, 10 to 50 mL of ethanol is added per 1 g of hyaluronic acid or its water-soluble salt. In step (4) above, the amount of water to be added is characterized by adding 0.5 to 2 mL of water per 1 g of hyaluronic acid or its water-soluble salt. The method according to any one of claims 4 to 6.

8. A composition containing a compound of formula I as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

9. The composition is characterized by further comprising a pharmaceutically acceptable carrier. The composition according to claim 8.

10. Use of a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition according to claim 8, in the manufacture of an anti-infective agent.

11. The aforementioned infection is characterized by being selected from infections caused by coronavirus (COVID-19). The use described in claim 10.

12. A method for anti-infectiveness, comprising administering to a patient a therapeutically effective amount of a compound of formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition according to claim 8.

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