A small molecule non-anticoagulant heparin and its preparation method and pharmaceutical composition and application

Abstract

The present invention discloses a small molecule non-anticoagulant heparin and its preparation method and its pharmaceutical composition and application. The preparation process of the small molecule non-anticoagulant heparin is simple and efficient, including: first preparing small molecule heparin by depolymerizing heparin, then oxidizing and destroying the heparin pentose structure site related to the anticoagulant activity of heparin with sodium periodate, and finally refining. Its molecular structure is clear, the weight-average molecular weight is 3000Da-5000Da, the reducing end of the sugar chain is mainly composed of 2,5-anhydro-D-mannose, and the middle of the sugar chain is still retained. The aldehyde group has low anticoagulant activity, but retains the biological activities of heparin such as anti-inflammatory, anti-allergic and anti-tumor, which can be applied to the application of heparin derivatives in the field of non-anticoagulant drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule non-anticoagulant heparin, a preparation method thereof, and a pharmaceutical composition and application thereof. Background Art

[0002] Heparin and low molecular weight heparin (LWMH) are widely used as anticoagulant and antithrombotic drugs in clinical practice. Their molecular structures are mainly composed of disaccharide repeating units of glucosamine (D-glucosamine, GlcN) and one of two uronic acids: iduronic acid (L-iduronic, IdoA) and glucuronic acid (D-glucuronic, GlcA), and sulfate groups are randomly modified at different positions of the sugar units. The anticoagulant activity of heparin mainly depends on binding with antithrombin-Ⅲ (AntithrombinⅢ, AT-Ⅲ) to produce anti-Xa (antithrombotic effect) and anti-Ⅱa active anticoagulant effects. The structure of heparin can be represented by a tetrasaccharide unit, and its structural formula is as follows: Figure 1 As shown. The pentasaccharide structure that binds to antithrombin is shown, Figure 2 As shown, it is the core structure necessary for maintaining anticoagulant activity.

[0003] In addition, the major part of the heparin sugar chain is the structurally ordered “regular region”—the structurally regular trisulfated disaccharide repeating unit—ΔUA2S-GlcNS6S (ΔIS), e.g. Figure 1 As shown in A; Figure 1 The low-sulfated region is shown in B, which is a disordered "irregular region" with a disordered structure, mainly containing some units containing IdoA and GlcA (followed by N-acetyl-GlcN or N-sulfonyl-GlcN (GlcNAc, GlcNS)) [RJ Linhardt, J. Med. Chem. 46, (2003) 2551-2564.], Figure 1 The content of B in the heparin oligosaccharide chain is relatively small. Figure 2 The pentasaccharide structure that binds to antithrombin is shown in the figure. The ones in bold are necessary to maintain the anticoagulant activity. If they are removed, the anticoagulant activity will decrease by 95%. The ones in italics are also very important. If they are removed, the anticoagulant activity will decrease by 25-50%. It is worth noting that in Figure 2 In the pentasaccharide structure, there is a special vicinal diol structure, which can be broken by strong oxidants.

[0004] Small molecule heparin is a relatively low molecular weight heparin prepared by chemical or enzymatic hydrolysis of unfractionated heparin (UFH), and its relative molecular weight is generally 3000-8000Da. It is well known that small molecule heparin has a lower anticoagulant effect (anti-IIa activity) than unfractionated heparin, but a stronger antithrombotic effect (anti-Xa activity), a reduced bleeding tendency, is safer and more effective, and has high bioavailability and a long half-life. Among the existing heparin anticoagulant and antithrombotic drugs, the anti-IIa activity and anti-Xa activity of unfractionated heparin are both above 180IU / mg, while the anti-Xa activity of various commercially available small molecule heparins is generally around 100IU / mg, but there are some differences in their anti-IIa activity, ranging from 15-55IU / mg, with an average of around 30IU / mg. In the field, it is generally believed that heparin with anti-IIa activity and anti-Xa activity below 10IU / mg is non-anticoagulant heparin.

[0005] Modern medical research has found that heparin and small molecule heparin and their derivatives have multiple biological functions such as anti-inflammatory, anti-allergic, anti-tumor, antiviral and lipid-lowering, and have become a hot topic of research in this field both at home and abroad. However, in clinical applications, due to the strong anticoagulant and antithrombotic activity of these heparin drugs, the amount of drugs used is limited, making it difficult to achieve the effective dose of the above biological activity. Second, the high anticoagulant activity is prone to cause side effects such as bleeding, making it impossible to play a role in the above biological function areas such as anti-inflammatory. Summary of the invention

[0006] The technical problem to be solved by the present invention is: to provide a small molecule non-anticoagulant heparin, a preparation method of small molecule non-anticoagulant heparin, and a pharmaceutical composition containing small molecule non-anticoagulant heparin. The small molecule non-anticoagulant heparin and the pharmaceutical composition can be used to prepare drugs for preventing and treating inflammation, allergies and tumors, viruses, and hyperlipidemia.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is: the small molecule non-anticoagulant heparin has the following structural formula:

[0008]

[0009] in,

[0010] R1=H or SO3Na;

[0011] R2=SO3Na or COCH3;

[0012] n=0-24.

[0013] The small molecule non-anticoagulant heparin has the following structural characteristics:

[0014] (1) Weight average molecular weight is 3000Da-5000Da;

[0015] (2) Oxidative ring opening at the 3,4-position of the non-reducing end to form a dialdehyde structure;

[0016] (3) The reducing end is mainly composed of 2,5-anhydro-D-mannose;

[0017] (4) An aldehyde group is retained in the middle of the sugar chain.

[0018] The small molecule non-anticoagulant heparin has an anti-Xa factor potency of less than 10 IU / mg in terms of pharmacology and pharmacodynamics.

[0019] The preparation method of the above-mentioned small molecule non-anticoagulant heparin, the schematic diagram of the synthetic route is as follows Figure 3 As shown, the following steps are included:

[0020] S1, nitrous acid method to depolymerize heparin to prepare small molecular weight heparin;

[0021] S2, periodate oxidation method destroys the anticoagulant active site of small molecular weight heparin;

[0022] S3. Refining.

[0023] The mechanism of action of nitrous acid degradation method for producing small molecular weight heparin is that under the action of low pH value, nitrous acid reacts with N-sulfated glucosamine in heparin to remove HSO4- to form -NH2, which then undergoes diazotization reaction with nitrous acid, releasing N2 while the glycosidic bond breaks, electrons are transferred, and the ring shrinks to form 2,5-anhydromannose.

[0024] Preferably, the preparation method of preparing small molecular heparin by depolymerizing heparin by nitrous acid method in step S1 of the present scheme is: dissolving heparin in water to prepare a first solution with a final concentration of 10-20%, adjusting the pH value of the first solution to 2-4 with acid, adding sodium nitrite in an amount of 0.02-0.05 times the weight of heparin, reacting at room temperature for 2-5 hours to obtain a first reaction solution, and the room temperature is 15-35°C; then adjusting the pH value to neutral, adding sodium chloride solid in an amount of 0.05-0.1 times the total volume of the first reaction solution, stirring to dissolve, and then adding ethanol or methanol in an amount of 1.0-2.5 times the total volume of the first reaction solution for precipitation, collecting the precipitate, and drying to obtain a solid reaction product.

[0025] The molecular weight of the solid reaction product of heparin depolymerization obtained in step S1 is controlled to be 5000 Da or less. The molecular weight of the reaction product varies depending on factors such as the amount of sodium nitrite used in the depolymerization reaction, pH value, temperature, time and alcohol precipitation conditions.

[0026] The solid reaction product after heparin depolymerization obtained in step S1 is small molecule heparin, and the reducing end of the main oligosaccharide chain is a 2,5-anhydro-D-mannose structure formed by ring contraction.

[0027] The mechanism of action of periodate oxidation to remove the anticoagulant activity of small molecular weight heparin is that under the action of sodium periodate, the sugar chain, especially the vicinal diol structure contained in the heparin pentose located at the active site, is oxidized. This vicinal diol carbon chain is broken to generate aldehyde. The reaction mechanism is as follows Figure 4 shown.

[0028] Preferably, in step S2 of the present scheme, the method for destroying the anticoagulant active sites of small molecule heparin by periodate oxidation is as follows: the reaction product obtained in step S1 is dissolved in water to prepare a second solution with a final concentration of 10-20%, the pH value of the second solution is adjusted to 4-6 with acid, and a 0.1 mol / L sodium periodate solution of equal volume to the second solution is added, and the reaction is carried out in a cool place away from light for not less than 16 hours to obtain a second reaction solution, and the temperature in the cool place is 10-20°C; then the pH is adjusted to neutral, 0.05-0.1 times the total volume of sodium chloride solid is added to the second reaction solution, and the solution is stirred to dissolve, and then 1.0-3.0 times the total volume of the second reaction solution is added to ethanol or methanol for precipitation, and the precipitate is collected.

[0029] Preferably, the reaction in step S2 of the preparation method needs to be protected from light to avoid photodecomposition of periodic acid.

[0030] The crude small molecule non-anticoagulant heparin is obtained by step S2. The vicinal diol structure contained in the sugar chain, especially in the heparin pentose located at the active site, is oxidized to break and generate aldehydes. In addition, the periodate oxidation method causes the 3,4-position vicinal diol carbon chain of the non-reducing end 2-sulfate-iduronic acid of the small molecule heparin prepared by step S1 to undergo oxidation and break and open the ring to form a dialdehyde structure, such as Figure 5 Preferably, the refining is specifically as follows: decolorization by oxidation with hydrogen peroxide or adsorption with activated carbon, removal of particulate matter by filtering, removal of impurities by precipitation with an organic solvent: methanol and / or ethanol, and removal of moisture by vacuum drying or freeze drying to obtain a small molecule non-anticoagulant heparin product.

[0031] The small molecule non-anticoagulant heparin has its anticoagulant and antithrombotic activities removed (the active center necessary for the anticoagulant and antithrombotic functions, the special vicinal diol structure of heparin pentose, is interrupted by sodium periodate oxidation), but retains other biological activities such as anti-inflammatory, anti-allergic, anti-tumor, antiviral, and lipid-lowering activities.

[0032] A pharmaceutical composition, comprising: a pharmaceutically acceptable carrier, and a small molecule non-anticoagulant heparin described in this scheme. The pharmaceutical composition can be used to prepare drugs for preventing and treating inflammation, allergies and tumors, viruses, hyperlipidemia, etc. In addition, the small molecule non-anticoagulant heparin described in this scheme can be used as a raw material to prepare drugs for preventing and treating inflammation, allergies and tumors, viruses, hyperlipidemia, etc.

[0033] The beneficial effects of the present invention are: providing a new type of small molecule non-anticoagulant heparin, a preparation method of small molecule non-anticoagulant heparin, and an application of small molecule non-anticoagulant heparin. The preparation method of the small molecule non-anticoagulant heparin is simple and efficient, has a unique molecular structure and modification, and a weight-average molecular weight of 3000Da-5000Da. The biological anticoagulant activity of (heparin) is removed, and its anti-Xa activity is no more than 10IU / mg, while its biological activities such as anti-inflammatory, anti-allergic and anti-tumor are fully retained. It is suitable for the development and preparation of drugs for preventing and treating inflammation, allergies and tumors, viruses, and hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The structure of heparin described in the background art can be represented by a structural formula using a tetrasaccharide unit. Figure 2 This is the structural formula showing the pentasaccharide structure that binds to antithrombin as described in the Background Art.

[0035] Figure 3 It is a schematic diagram of the synthetic route for the preparation of small molecule non-anticoagulant heparin.

[0036] Figure 4 This is a reaction mechanism diagram of the mechanism of action of periodate oxidation method to remove the anticoagulant activity of small molecular weight heparin.

[0037] Figure 5 This is a schematic diagram of the periodate oxidation method causing the 3,4-position vicinal diol carbon chain of the non-reducing end 2-sulfate-iduronic acid of the small molecule heparin prepared in step S1 to undergo oxidative cleavage and ring opening to form a dialdehyde structure.

[0038] Figure 6 It is the NMR spectrum of non-anticoagulant heparin ( 1 H-NMR) schematic diagram.

[0039] Figure 7 It is a schematic diagram of the molecular weight and molecular weight distribution of small molecule non-anticoagulant heparin.

[0040] Figure 8 is the level of TNF-α in the supernatant of BV-2 cells stimulated with LPS for 24 h after treatment with small molecule non-anticoagulant heparin.

[0041] Fig. 9is the level of IL-6 in the supernatant of BV-2 cells stimulated with LPS for 24 h after treatment with small molecule non-anticoagulant heparin.

[0042] Fig.10 is the level of IL-4 in the supernatant of BV-2 cells stimulated with LPS for 24 hours after treatment with small molecule non-anticoagulant heparin. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0044] Embodiment 1

[0045] The small molecule non-anticoagulant heparin described in the present invention has the following structural formula:

[0046]

[0047] in,

[0048] R1=H or SO3Na;

[0049] R2=SO3Na or COCH3;

[0050] n=0-24.

[0051] The small molecule non-anticoagulant heparin has the following structural characteristics:

[0052] (1) Weight average molecular weight is 3000Da-5000Da;

[0053] (2) Oxidative ring opening at the 3,4-position of the non-reducing end to form a dialdehyde structure;

[0054] (3) The reducing end is mainly composed of 2,5-anhydro-D-mannose;

[0055] (4) An aldehyde group is retained in the middle of the sugar chain.

[0056] The small molecule non-anticoagulant heparin has an anti-Xa factor potency of less than 10 IU / mg in terms of pharmacology and pharmacodynamics.

[0057] The above-mentioned small molecule non-anticoagulant heparin can be used to prepare drugs for preventing and treating inflammation, allergy, tumor, virus and hyperlipidemia.

[0058] Embodiment 2

[0059] A pharmaceutical composition described in this embodiment includes: a pharmaceutically acceptable carrier and the small molecule non-anticoagulant heparin described in Example 1. The pharmaceutically acceptable carrier includes: a pH buffer, an isotonicity regulator, a preservative, an excipient, etc., which are clearly defined in conventional pharmaceutical preparations.

[0060] The above-mentioned pharmaceutical composition can be used to prepare drugs for preventing and treating inflammation, allergy, tumor, virus and hyperlipidemia.

[0061] Embodiment 3

[0062] The preparation of the small molecule non-anticoagulant heparin described in this embodiment is based on Figure 3 The schematic diagram of the synthetic route shown in the figure is to prepare small molecule non-anticoagulant heparin, and the steps are as follows:

[0063] (1) Depolymerization: 100 g of heparin sodium was placed in a 1 L three-necked flask, 500 mL of water was added and stirred until dissolved, the pH was adjusted to 2.0 with dilute hydrochloric acid, the mixture was placed in a 35°C water bath and stirred, and a sodium nitrite solution was added, wherein the sodium nitrite solution was formed by dissolving 2 g of sodium nitrite in about 5 mL of water, and the reaction began. After 5 hours, the pH was adjusted to neutral with a dilute alkali solution to terminate the reaction; the mixture was transferred to a beaker, 6 g of sodium chloride was added, stirred to dissolve, and 600 mL of ethanol was added. The mixture was stirred thoroughly and allowed to stand overnight; the supernatant was discarded, and the precipitate was dehydrated with 300 mL of ethanol and dried under vacuum to obtain a total of 87 g of depolymerized small molecular heparin;

[0064] (2) Oxidation: 85 g of depolymerized small molecular weight heparin was placed in a 2 L three-necked flask, 450 mL of water was added and stirred until dissolved, the pH was adjusted to 4.0 with dilute hydrochloric acid, cooled and kept warm in a water bath at 10°C, 450 mL of 0.1 mol / L sodium periodate solution was added, protected from light, stirred to start the reaction, and the pH was adjusted to neutral after 16 hours to terminate the reaction; the mixture was transferred to a beaker, 45 g of sodium chloride was added, stirred to dissolve, and then 900 mL of ethanol was added, stirred thoroughly and allowed to stand overnight; the supernatant was discarded and the precipitate was collected;

[0065] (3) Purification: add 450 mL of 5% sodium chloride solution to the precipitate, stir until dissolved, add 30 mL of 30% hydrogen peroxide solution, stir and stand at room temperature for decolorization, the reaction solution turns light yellow after 2 hours; adjust the pH of the reaction solution to neutral, filter with 0.45 μm + 0.22 μm filter membrane, add 500 mL of ethanol, stir well and stand overnight; discard the supernatant, collect the precipitate, add 300 mL of ethanol to pulp and dehydrate, collect the precipitate, and vacuum dry to obtain a small molecule non-anticoagulant heparin, a total of 63 g, with a total yield of 63%.

[0066] The product test results are as follows: the loss on drying is 5.2%, the weight average molecular weight is 4200Da, the anti-Xa activity after dryness is 5.3U / mg, and the anti-IIa activity after dryness is 2.2U / mg.

[0067] Embodiment 4

[0068] The preparation of the small molecule non-anticoagulant heparin described in this embodiment is based on Figure 3 The schematic diagram of the synthetic route shown in the figure is to prepare small molecule non-anticoagulant heparin. The experimental steps are as follows:

[0069] (1) Depolymerization: 100 g of heparin sodium was placed in a 2 L three-necked flask, 1000 mL of water was added and stirred until dissolved, dilute hydrochloric acid was used to adjust the pH to 4.0, and the mixture was stirred at 15°C. A sodium nitrite solution was added, wherein the sodium nitrite solution was prepared by dissolving 5 g of sodium nitrite in about 10 mL of water. The reaction began, and after 2 hours, the pH was adjusted to neutral with a dilute alkali solution to terminate the reaction. The mixture was transferred to a beaker, 100 g of sodium chloride was added, and the mixture was stirred until dissolved. 1650 mL of methanol was added, and the mixture was stirred thoroughly and allowed to stand for 3 hours. The supernatant was discarded, and the precipitate was dehydrated with 300 mL of methanol and dried under vacuum to obtain a total of 89 g of depolymerized small molecular weight heparin.

[0070] (2) Oxidation: 85 g of depolymerized small molecular weight heparin was placed in a 2 L three-necked flask, 850 mL of water was added and stirred until dissolved, the pH was adjusted to 6.0 with dilute hydrochloric acid, and the mixture was placed in a 25°C water bath, 850 mL of 0.1 mol / L sodium periodate solution was added, and the reaction was started by stirring. The mixture was protected from light and stirred for 20 hours, and the pH was adjusted to neutral to terminate the reaction; the mixture was transferred to a beaker, 170 g of sodium chloride was added, and stirred to dissolve, and then 5000 mL of methanol was added. The mixture was stirred thoroughly and allowed to stand for 3 hours; the supernatant was discarded and the precipitate was collected;

[0071] (3) Purification: add 850 mL of 10% sodium chloride solution to the precipitate, stir until dissolved, add 50 mL of 30% hydrogen peroxide solution, stir and stand at room temperature for decolorization, the reaction solution turns light yellow after 3 hours; adjust the pH of the reaction solution to neutral, filter with 0.22 μm filter membrane, add 850 mL of methanol, stir thoroughly and stand overnight; discard the supernatant, collect the precipitate, add 400 mL of methanol, beat and dehydrate, collect the precipitate, and vacuum dry to obtain small molecule non-anticoagulant heparin, a total of 68 g, with a total yield of 68%.

[0072] The product test results are as follows: the loss on drying is 4.8%, the weight average molecular weight is 3850Da, the anti-Xa activity after dryness is 4.8U / mg, and the anti-IIa activity after dryness is 2.3U / mg.

[0073] Embodiment 5

[0074] The preparation of the small molecule non-anticoagulant heparin described in this embodiment is based on Figure 3 The schematic diagram of the synthetic route shown in the figure is to prepare small molecule non-anticoagulant heparin. The experimental steps are as follows:

[0075] (1) Depolymerization: 100 g of sodium heparin was placed in a 2L three-necked flask, 1000 mL of water was added and stirred until dissolved, the pH was adjusted to 3.0 with dilute hydrochloric acid, and the mixture was placed in a 20°C water bath, and a sodium nitrite solution was added, wherein the sodium nitrite solution was formed by dissolving 3 g of sodium nitrite in about 8 mL of water. The reaction started, and after 3 hours, the pH was adjusted to neutral with a dilute alkali solution to terminate the reaction; 110 g of sodium chloride was added, stirred until dissolved, the solution was transferred to a beaker, and 2200 mL of methanol was added, stirred thoroughly, and then allowed to stand overnight; the supernatant was discarded, and the precipitate was dehydrated with 200 mL of methanol and dried in vacuo to obtain a total of 92 g of depolymerized small molecular heparin;

[0076] (2) Oxidation: 85 g of depolymerized small molecular weight heparin was placed in a 2 L three-necked flask, 850 mL of water was added and stirred until dissolved, the pH was adjusted to 5.0 with dilute hydrochloric acid, cooled and kept warm in a water bath at 15°C, 850 mL of 0.1 mol / L sodium periodate solution was added, protected from light, and the reaction was started. After stirring for 20 hours, the pH was adjusted to neutral to terminate the reaction; 170 g of sodium chloride was added, stirred to dissolve, and then about 3500 mL of methanol was added. After sufficient stirring, the mixture was allowed to stand overnight; the supernatant was discarded and the precipitate was collected;

[0077] (3) Purification: add 850 mL of 3% sodium chloride solution to the precipitate, stir until dissolved, add 50 mL of 30% hydrogen peroxide solution, stir and stand at room temperature for decolorization, and the reaction solution turns light yellow after 4 hours; adjust the pH of the reaction solution to neutral, filter with a 0.45 μm + 0.22 μm filter membrane, add 1700 mL of methanol, stir thoroughly and stand overnight; discard the supernatant, collect the precipitate, add 400 mL of methanol to pulp and dehydrate, collect the precipitate, and vacuum dry to obtain a small molecule non-anticoagulant heparin, a total of 71 g, with a total yield of 71%.

[0078] The product test results are as follows: the loss on drying is 4.5%, the weight average molecular weight is 3950Da, the anti-Xa activity after dryness is 4.1U / mg, and the anti-IIa activity after dryness is 1.7U / mg.

[0079] From Example 3, Example 4, and Example 5, we can see that the product test results show that the anti-Xa factor activity of the small molecule non-anticoagulant heparin is below 10 IU / mg, and the anti-IIa factor activity is below 5 IU / mg.

[0080] Embodiment 6

[0081] The kilogram-scale preparation of the small molecule non-anticoagulant heparin described in this embodiment is based on Figure 3 The schematic diagram of the synthetic route shown in the figure is to prepare small molecule non-anticoagulant heparin. The experimental steps are as follows:

[0082] (1) Depolymerization: 3.0 kg of heparin sodium and 30 L of purified water were added to a 100 L reactor, and the mixture was stirred at 180 rpm until the mixture was clear. The reaction temperature was controlled to 20° C. The pH of the reaction solution was adjusted to 3.0 with dilute hydrochloric acid, and a sodium nitrite solution was added, wherein the sodium nitrite solution was formed by dissolving 90 g of sodium nitrite in about 300 mL of water. After the reaction started, the pH was adjusted to 7 with a dilute alkali solution after 3 hours to terminate the reaction. 3.3 kg of sodium chloride was added and stirred until the mixture was clear. The reaction solution was transferred to an alcohol precipitation kettle, and 66 L of methanol was added. After sufficient stirring, the mixture was allowed to stand overnight. The supernatant was discarded, and the precipitate was dehydrated with about 6 L of methanol. The wet precipitate was vacuum dried to obtain a total of 2.65 kg of depolymerized small molecular weight heparin.

[0083] (2) Oxidation: Take 2.5 kg of depolymerized small molecular heparin and add it to a 150 L reactor (wrapped in a dark container), stir with 25 L of purified water at 180 rpm until dissolved, control the temperature in a cold water bath to 15°C, adjust the pH to 5.0 with dilute hydrochloric acid, and start the reaction by adding 25 L of 0.1 mol / L sodium periodate solution. After 20 hours, adjust the pH to 7.1, add 5.0 kg of sodium chloride, stir until dissolved, transfer the reaction solution to an alcohol precipitation kettle, add 110 L of methanol, stir thoroughly, let stand overnight, discard the supernatant, and collect the precipitate;

[0084] (3) Purification: Add 25 L of 3% sodium chloride solution to the precipitation tank, stir until dissolved, add 1.5 L of 30% hydrogen peroxide solution, stir and stand at room temperature for 4 hours to decolorize; adjust the pH of the reaction solution to neutral, add 150 g of activated carbon, stir at 180 rpm for 15 minutes, filter with titanium rod to remove carbon residue, filter the filtrate with 0.45 μm and 0.22 μm filters respectively, add 50 L of methanol to the clear liquid, stir well and stand overnight; discard the supernatant, collect the precipitate, add about 10 L of methanol to pulp and dehydrate, The precipitate was collected and dried; then dissolved in 10 L of water for injection, filtered at 0.22 μm, loaded into a stainless steel freeze-drying tray, and freeze-dried (procedure: pre-freeze at -40°C for 4 hours, start the cold trap to cool down and vacuum pump (50 Pa)), the partition was raised to 0°C for 2 hours, maintained at 0°C for 12 hours, and vacuumed at 20±5 Pa; the partition was raised to 40°C for 2 hours, maintained at 40°C for 2 hours, and vacuumed to the limit; after completion, the vacuum was released and the dish was taken out of the box), and a total of 1.89 kg of small molecule non-anticoagulant heparin was obtained with a yield of 63%.

[0085] The test results of the product are as follows: the loss on drying is 1.2%. The weight average molecular weight is 4050Da; the anti-Xa activity is 4.3U / mg after dryness; the anti-IIa activity is 2.7U / mg after dryness; the solution of 1.0g product dissolved in 10mL water is clear and the color is not darker than the standard color No. 6; the solution of 1.0g product dissolved in 10mL water has a pH of 6.9; the sodium content is 12.2% after dryness; the sulfonate / carboxylate ratio is 2.2; the heavy metal residue is not more than 20ppm; the methanol in the solvent residue is 431ppm; the bacterial endotoxin content is less than 0.1EU / mg.

[0086] Batch inspection of small molecule non-anticoagulant heparin: Table 1 shows the quality inspection results of three consecutive batches of 5 kg scale-up validation batches. The preparation method is the same as that of Example 6, except for the batch size and some detailed process parameters.

[0087] Table 1. Test results of three consecutive batches of 5Kg small molecule non-anticoagulant heparin

[0088]

[0089]

[0090] The above data show that the quality inspection results of the three batches of small molecule non-anticoagulant heparin in production verification are highly consistent, which indirectly reflects the controllability of the preparation process and quality. From the above data, it can be concluded that the anti-Xa factor activity of small molecule non-anticoagulant heparin is below 10IU / mg, and the anti-IIa factor activity is below 5IU / mg.

[0091] H NMR spectrum of small molecule non-anticoagulant heparin ( 1 H-NMR) analysis: The small molecule non-anticoagulant heparin sample prepared in Example 6 1 H-NMR analysis experiments were used to determine its structural characteristics.

[0092] Test compound: small molecule non-anticoagulant heparin sample (Batch No.: 20221001).

[0093] Sample preparation: 35 mg of sample was dissolved in 0.6 mL of deuterated water (0.002% TSP was added as internal standard to correct the zero point), the solution was transferred to a 5 mm NMR tube, and ultrasonicated for 2 minutes.

[0094] Instrument: NMR Bruker AC500HD;

[0095] Software: Bruker Top Spin 3.2 (Bruker Biospin GmbH);

[0096] result: 1 H-NMR spectrum is shown in Figure 6 .

[0097] like Figure 6 As shown, the small molecule non-anticoagulant heparin sample has H1 of GlcNAc6S / GlcNS6S, H1 of IdoA2S, H2 of GlcNS, and -CH3 hydrogen peak (methyl hydrogen) on the acetyl group of GlcNAc at 5.42ppm, 5.21ppm, 3.28ppm (double peak) and 2.05ppm, respectively, which shows that the main structure of the small molecule non-anticoagulant heparin is consistent with that of heparin polysaccharide. In addition, there are multiple groups of aldehyde signals in the low field region of 10ppm-8.5ppm, which corresponds to the chemical reaction mechanism of this product and also reflects the existence of aldehyde structure in its oligosaccharide chain.

[0098] Molecular weight distribution (SEC-HPLC) analysis of small molecule non-anticoagulant heparin: The molecular weight distribution (SEC-HPLC) analysis experiment of the small molecule non-anticoagulant heparin sample prepared in Example 6 was used to determine its structural characteristics.

[0099] (1) Equipment and reagents:

[0100] Equipment: Agilent 1100 high performance liquid chromatograph, Agilent G1362A differential refractometer, electronic balance;

[0101] Molecular weight calibrants: USP standard substances;

[0102] Reagents: lithium nitrate (Sinopharm, AR), ultrapure water;

[0103] Test compound: small molecule non-anticoagulant heparin sample (Batch No.: 20221001).

[0104] (2) Testing conditions:

[0105] According to the high performance liquid chromatography analysis method, TSKgel Guard Column SWXL, TSKgel G3000SWXL (7.8mm×30cm, 5μm) and TSKgel G2000SWXL (7.8mm×30cm, 5μm) chromatographic columns were selected and connected in series, and detected by a differential refractive index detector. The detection conditions were as follows:

[0106] HPLC mobile phase: 0.5 M lithium nitrate, pH 6.5;

[0107] Flow rate: 0.6 mL / min;

[0108] Detection time: 50min;

[0109] Column temperature: 30°C;

[0110] Reference cell and detection cell temperature: 30°C;

[0111] Injection volume: 20 μL;

[0112] Preparation of HPLC mobile phase: Weigh 34.5 g lithium nitrate, place in a 1 L volumetric flask, dissolve in 700 mL ultrapure water, adjust pH to 6.5, dilute to scale with ultrapure water, shake well, and filter through a 0.22 μm microporous membrane;

[0113] Test results: SEC-HPLC analysis and GPC software were used to calculate the molecular weight and molecular weight distribution of small molecule non-anticoagulant heparin. The results are shown in Table 2 and Figure 7 .

[0114] Table 2. Molecular weight and molecular weight distribution results of small molecule non-anticoagulant heparin

[0115]

[0116] Note: Mw is the weight average molecular weight, Mn is the number average molecular weight, 8000 is the percentage of components with molecular weight greater than 8000Da, M 2000-8000 is the percentage of components with molecular weight between 2000Da and 8000Da, M 2000 is the percentage of components with molecular weight less than 2000Da, Pd is the dispersion = Mw / Mn; M max is the maximum molecular weight.

[0117] From the results, we can see that the weight average molecular weight of small molecule non-anticoagulant heparin is about 4000Da, and the molecular weight is greater than 8000Da component (M 8000 ) percentage is less than 10%, and the molecular weight is between 2000Da and 8000Da components (M 2000-8000 ) percentage is between 70% and 85%, and the molecular weight is less than 2000Da components (M 2000 ) is less than 20%. In addition, according to the above, the sulfate substitution degree (sulfonate / carboxylate ratio) of small molecule non-anticoagulant heparin is 2.2, so the molecular weight of a disaccharide unit is about 550Da, and the maximum value of the number of repeating disaccharide units n in the structural formula of the small molecule non-anticoagulant heparin is 24, calculated based on the maximum molecular weight of 15195Da.

[0118] This experiment verified the effect of small molecule non-anticoagulant heparin on LPS-induced neuroinflammation in BV-2 cells. Lipopolysaccharide (LPS) was used to stimulate microglial BV-2 cells to induce neuroinflammation. Different concentrations of small molecule non-anticoagulant heparin (500μM, 25μM and 1μM), positive control (dexamethasone) and saline blank control were set up for treatment. All groups were repeated 6 times, and the cell culture supernatant was collected and the cytokine levels were evaluated by electrochemiluminescence (MSD) assay.

[0119] The experiment is as follows:

[0120] Mouse microglial cells BV-2 were cultured in Opti-MEM medium containing 10% fetal calf serum (FCS), 1% penicillin / streptomycin and 2mM L-glutamine, counted, and seeded into 96-well plates, with 5000 BV-2 cells per well. After 48 hours, the medium was replaced with treatment medium (DMEM medium containing 5% FCS, 2mM L-glutamine) and cultured continuously.

[0121] One hour before LPS stimulation, different concentrations of small molecule non-anticoagulant heparin (500μM, 25μM and 1μM), positive control (LPS+positive drug (dexamethasone 10μM)), saline blank control and control group treated with LPS alone were treated in the same way. 24 hours after LPS induction, BV-2 cell supernatant was collected and diluted 1-fold. Three cytokines were determined by immunoadsorption according to the instructions of the ELISA kit: pro-inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); anti-inflammatory cytokine: interleukin-4 (IL-4), and the results were expressed in pg / mL.

[0122] Experimental results:

[0123] Figure 8 , Fig. 9 , Fig.10 The levels of TNF-α, IL-6 and IL-4 in the supernatant of BV-2 cells after LPS stimulation for 24 hours after treatment with small molecule non-anticoagulant heparin, respectively. BV-2 cells were treated with 3 concentrations of small molecule non-anticoagulant heparin for 24 hours, and the release levels of cytokines in the supernatant were evaluated by MSD; data units are pg / mL and displayed as bar graphs, Mean±SEM (N=6 per group); VC: saline control, TI: small molecule non-anticoagulant heparin, RI: positive control; One-way ANOVA was performed with the control group stimulated with LPS, and the differences in data between groups were tested by One way Anova, **p<0.01, ***p<0.001.

[0124] After 24 hours of LPS stimulation, the levels of cytokines TNF-α and IL-6 increased significantly. On the contrary, the level of IL-4 decreased significantly after LPS stimulation. In the positive control group, the levels of TNF-α and IL-6 decreased due to the use of the positive drug dexamethasone.

[0125] Compared with the control group treated with LPS alone, the three groups of small molecule non-anticoagulant heparin with different concentrations significantly reduced TNF-α cytokines in a dose-dependent manner. The higher the dose, the better the effect. Figure 8 As shown in Figure 2, the level of IL-6 was significantly reduced in the 500μM and 25μM groups compared with the LPS control group, but had no effect in the low concentration 1μM group. Fig. 9 As shown. Compared with the LPS control, the anti-inflammatory cytokine IL-4, BV-2 cells treated with high and medium concentration groups (500μM and 25μM) of small molecule non-anticoagulant heparin showed a significant increase in IL-4 levels, as shown Fig.10 shown.

[0126] The experimental results show that lipopolysaccharide (LPS) is used to stimulate microglial cells BV-2 to induce neuroinflammation. When treated with small molecule non-anticoagulant heparin, it can significantly inhibit the release of cellular inflammatory factors TNF-α and IL-6, and increase the concentration of anti-inflammatory factor IL-4, thereby achieving an anti-inflammatory effect.

[0127] Finally, it should be noted that the above is only a preferred embodiment of the present invention. It should be pointed out that the protection scope of the present invention is not limited to the description scope of the above embodiments. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A small molecule non-anticoagulant heparin, characterized in that: Its structural formula is as follows: in, R1=H or SO3Na. R2=SO3Na or COCH3; n＝0-24； The weight average molecular weight is 3000Da-5000Da; the non-reducing end is oxidized at the 3,4-position to form a dialdehyde structure; the reducing end is mainly composed of 2,5-anhydro-D-mannose; the aldehyde group is retained in the middle of the sugar chain; The anti-factor Xa titer was below 10 IU / mg.

2. A method for preparing a small molecule non-anticoagulant heparin according to claim 1, characterized in that: The steps include: S1, nitrous acid method to depolymerize heparin to prepare small molecular weight heparin; The preparation method of preparing small molecular heparin by depolymerizing heparin by nitrous acid method is as follows: dissolving heparin in water to prepare a first solution with a final concentration of 10-20%, adjusting the pH value of the first solution to 2-4 with acid, adding sodium nitrite in an amount of 0.02-0.05 times the weight of heparin, reacting at room temperature for 2-5 hours to obtain a first reaction solution, the room temperature being 15-35°C; then adjusting the pH value to neutral, adding sodium chloride solid in an amount of 0.05-0.1 times the total volume of the first reaction solution, stirring to dissolve, then adding ethanol or methanol in an amount of 1.0-2.5 times the total volume of the first reaction solution for precipitation, collecting the precipitate, and drying to obtain a reaction product; S2, periodate oxidation method destroys the anticoagulant active site of small molecular weight heparin; The method of destroying the anticoagulant active site of small molecular heparin by periodate oxidation is as follows: the reaction product obtained in step S1 is dissolved in water to prepare a second solution with a final concentration of 10-20%, the pH value of the second solution is adjusted to 4-6 with acid, and a 0.1 mol / L sodium periodate solution of equal volume to the second solution is added, and the reaction is carried out in a cool place away from light for not less than 16 hours to obtain a second reaction solution, and the temperature in the cool place is 10-20°C; then the pH is adjusted to neutral, 0.05-0.1 times the total volume of sodium chloride solid is added, and the solution is dissolved by stirring, and then 1.0-3.0 times the total volume of the second reaction solution is added with ethanol or methanol for precipitation, and the precipitate is collected; S3, refining; The refining process includes: decolorizing by oxidation with hydrogen peroxide or adsorption with activated carbon, filtering out particulate matter by a filter, removing impurities by precipitation with methanol and / or ethanol, and removing moisture by vacuum drying or freeze drying to obtain a small molecule non-anticoagulant heparin product.

3. The method for preparing a small molecule non-anticoagulant heparin according to claim 2, characterized in that: In step S1, the molecular weight of the reaction product obtained by depolymerizing heparin by the nitrous acid method to prepare small molecular weight heparin is controlled to be 5000 Da or less.

4. A pharmaceutical composition, characterized in that: include: A pharmaceutically acceptable carrier, and a small molecule non-anticoagulant heparin according to claim 1.

5. Use of a small molecule non-anticoagulant heparin as claimed in claim 1 as a raw material in the preparation of drugs for preventing and treating inflammation, allergy and tumor, virus and hyperlipidemia.

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