Macromolecular pharmaceutical composition based on deep eutectic solvent technology and preparation method

Abstract

The invention discloses a deep eutectic solvent which is an organic molten salt consisting of an anion component and a cation component, the anion component is selected from organic acid, polyhydric alcohol or sugar which can be used as a hydrogen bond donor; the cationic component is selected from quaternary ammonium salts and zwitterionic surfactants which can be used as hydrogen bond acceptors; and the molar ratio of the cation component to the anion component is 1: 4-1: 0.5. The invention further discloses a macromolecular pharmaceutical composition prepared on the basis of the deep eutectic solvent technology. The macromolecular pharmaceutical composition comprises an external enteric-coated protective layer and drug-loaded composite particles or drug-loaded composite pellets packaged in the enteric-coated protective layer, the drug-loaded composite particles or drug-loaded composite pellets are prepared from a deep eutectic solvent-macromolecular drug delivery system and other auxiliary materials; the deep eutectic solvent-macromolecular drug delivery system is prepared from a biological macromolecular drug and a deep eutectic solvent. According to the drug delivery system, the intestinal permeability of macromolecular drugs can be enhanced, and the oral bioavailability of the macromolecular drugs is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations and relates to a macromolecular drug composition and preparation method based on deep eutectic solvent technology, particularly to a safe and universally applicable oral delivery formulation of macromolecular drugs and its preparation method. Background Technology

[0002] Oral administration is the preferred route of drug delivery in clinical practice due to its high safety and good patient compliance. Large molecule drugs such as proteins, peptides, sugars or polysaccharides, and enzymes have advantages such as high therapeutic specificity, low toxicity and side effects, strong efficacy, and low accumulation in the body, making them widely used in the treatment of clinical diseases. However, large molecule drugs have drawbacks such as large molecular weight, poor membrane permeability, and instability due to the influence of digestive juices. Furthermore, various physiological barriers in the gastrointestinal tract, such as pH, enzyme barriers, mucus barriers, and intestinal epithelial barriers, also limit the oral absorption of large molecule drugs. The oral bioavailability of large molecule drugs is typically less than 1%, making it difficult to achieve effective therapeutic blood concentrations. Currently, subcutaneous injection is the primary route of administration in clinical practice, and delivering large molecule drugs to the target site orally presents a significant challenge. Therefore, effectively improving oral bioavailability remains crucial for the development of oral formulations of large molecule drugs.

[0003] Currently, methods to improve the oral bioavailability of macromolecular drugs can be mainly divided into three categories: first, improving the stability of the drug in the gastrointestinal tract; second, enhancing the intestinal permeability of the drug; and third, promoting the lymphatic absorption of the drug. Among them, enhancing the intestinal permeability of the drug can significantly improve its oral bioavailability and is a widely used delivery strategy in clinical practice to improve the oral absorption of macromolecular drugs. Commonly used permeation-enhancing methods are mainly divided into the following three types: (1) Combining macromolecular drugs with lipid components (such as squalene, polymethyl methacrylate, etc.) to form nanoparticles or microparticles to improve the lipophilicity of the drug. The lipid components in the nanoparticles help to enhance the interaction between the intestinal epithelial cell membrane and the nanoparticles, promote the adsorption of intestinal epithelial cells and the endocytosis of nanoparticles, thereby improving the oral absorption of the drug. (2) Coupling macromolecular drugs or drug carriers with specific target molecules (such as bile salts, folic acid, vitamin B12, transferrin, etc.) to enhance the targeting of the drug. There are various binding transporters on the intestinal epithelial cell membrane. Coupled with specific target molecules, the affinity of macromolecular drugs or drug carriers to binding transporters can be enhanced, thereby improving the intestinal transport rate of drugs. (3) Use absorption enhancers (such as surfactants, bile salts, ethylenediaminetetraacetic acid, and chitosan and its derivatives) to improve intestinal permeability, thereby improving the absorption efficiency of drugs. The mechanism by which absorption enhancers improve the oral absorption of macromolecular drugs is that they can open the tight junctions between intestinal epithelial cells or increase cell membrane permeability. All of the above methods can effectively improve the intestinal permeability of macromolecular drugs. Among them, the strategy of using absorption enhancers to improve intestinal permeability is the most widely used, and there are already successful cases of market launch.

[0004] based on Semaglutide tablets (Rybelsus), developed using advanced technology to achieve oral absorption, are the first marketed oral formulation of a large-molecule drug. Their core technology lies in the use of sodium N-[8-(2-hydroxyphenyl)amino]octanoate (SNAC) as an absorption enhancer. SNAC can neutralize the acidic environment surrounding the tablet to some extent, inhibit pepsin activity, reduce drug degradation, and promote semaglutide absorption in the stomach via transcellular pathways. However, clinical data indicate that the oral bioavailability of semaglutide tablets is less than 1%, limiting its application. Octreotide oral capsules are based on transient permeability enhancers. The oral formulation developed using this technology is the first oral somatostatin analog used to treat acromegaly. The technology uses sodium octanoate (C8) as an absorption enhancer, and forms a lipophilic suspension with sodium octanoate, peptides, sodium octanoate and polyvinylpyrrolidone (PVP) in addition to a hydrophobic medium, which is then encapsulated in enteric-coated capsules for delivery. The technology can protect drugs from digestive enzymes and instantly open tight junctions in the intestinal epithelium, promoting intestinal absorption of drug molecules. However, its oral bioavailability is as low as 0.25%, and it has significant intestinal irritation. Based on peptide oral delivery... In terms of technology, Oramed has developed an enteric-coated insulin capsule (ORMD-0801). The technology utilizes the chelating agent EDTA as an absorption enhancer to open the tight junctions of intestinal epithelial cells, while adding a protease inhibitor to slow drug degradation in the intestine. This phase III clinical trial has been terminated. In summary, currently in clinical practice, only low-molecular-weight macromolecular drugs (molecular weight less than 5000D) are approved for oral administration, but oral bioavailability is low, and the successful cases mentioned above are difficult to apply to the oral delivery of other macromolecular drugs. Furthermore, technology, Technology and The absorption enhancers used in these technologies are all surfactants. While increasing the intestinal permeability of drugs, they can irreversibly disrupt the normal function of intestinal cell membranes and may also allow harmful substances such as bacteria and fungi to invade, causing adverse gastrointestinal reactions. Summary of the Invention

[0005] The purpose of this invention is to prepare a deep-eutectic solvent (DES) using choline and geraniol, natural carrier materials with good biocompatibility and high safety, and to construct a universal and easy-to-prepare drug delivery system based on the deep-eutectic solvent technology. This drug delivery system can enhance the intestinal permeability of macromolecular drugs, improve the oral bioavailability of macromolecular drugs, and does not exhibit intestinal toxicity. Furthermore, the drug delivery system is prepared into drug-loaded composite particles and encapsulated with an enteric protective layer to obtain a macromolecular drug composition suitable for oral administration.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A deep eutectic solvent, wherein the deep eutectic solvent is an organic molten salt composed of an anionic component and a cationic component; wherein the anionic component is selected from organic acids (such as geraniic acid), polyols or sugars that can act as hydrogen bond donors; the cationic component is selected from quaternary ammonium salts (such as choline or its bicarbonate) and amphoteric surfactants that can act as hydrogen bond acceptors; and the molar ratio of the cationic component to the anionic component is 1:4 to 1:0.5.

[0008] The melting point of the deep eutectic solvent is typically below 100°C.

[0009] Preferably, the anionic component is geraniol; and the cationic component is choline or its bicarbonate.

[0010] Preferably, the molar ratio of the cationic component to the anionic component is 1:4, 1:2, 1:1, 3:2, or 2:1.

[0011] More preferably, the molar ratio of the cationic component to the anionic component is 1:2.

[0012] The deep eutectic solvent is prepared by the following method: using water as the reaction solvent, the aqueous solution of the cationic component is placed in a water bath at 35℃~40℃, and the solution of the anionic component is added dropwise to the aqueous solution of the cationic component under stirring conditions until no carbon dioxide is released; after the reaction is completed, the solvent in the reaction solution is removed by vacuum evaporation, and the product is then dried in a vacuum drying oven to obtain the deep eutectic solvent.

[0013] A macromolecular drug composition prepared based on deep eutectic solvent technology includes an outer enteric protective layer and drug-loaded composite particles or drug-loaded composite microspheres encapsulated inside the enteric protective layer; the drug-loaded composite particles or drug-loaded composite microspheres are prepared by a deep eutectic solvent-macromolecular drug delivery system and other excipients; the deep eutectic solvent-macromolecular drug delivery system is made of a biological macromolecular drug and a deep eutectic solvent as described in this invention.

[0014] The macromolecular drug composition is administered orally, and the dosage form of the macromolecular drug composition is an oral formulation. Specifically, the oral dosage form includes enteric-coated capsules, enteric-coated tablets, enteric-coated granules, or enteric-coated microcapsules, etc.

[0015] The enteric protective layer is an enteric capsule shell or an enteric coating.

[0016] When the dosage form of the macromolecular pharmaceutical composition is an enteric-coated capsule, the enteric protective layer is a commercially available enteric-coated capsule shell.

[0017] When the macromolecular pharmaceutical composition is an enteric-coated tablet, enteric-coated microcapsule, or enteric-coated microparticle, the enteric protective layer is an enteric coating.

[0018] The aforementioned biological macromolecular drugs are drugs such as proteins, sugars or polysaccharides, polypeptides, and enzymes.

[0019] Preferably, the biopharmaceutical includes low molecular weight heparin, calcitonin, or uricase, and can be prepared into oral macromolecular drug compositions using a similar method based on a deep eutectic solvent.

[0020] The excipients include one or more components such as fillers, lubricants, and disintegrants.

[0021] The filler may be lactose, mannitol or sorbitol, preferably mannitol.

[0022] The lubricant is magnesium stearate.

[0023] The disintegrant is starch.

[0024] Another object of the present invention is to provide a method for preparing the macromolecular pharmaceutical composition described above, comprising:

[0025] Step (1): Place the aqueous solution of the cationic component in a water bath at 35℃~40℃. Under stirring conditions, add the aqueous solution of the anionic component dropwise to the cationic component solution and react until no carbon dioxide is released.

[0026] Step (2): After the reaction is complete, the solvent in the reaction solution is removed by vacuum evaporation.

[0027] Step (3): Place the product in a vacuum drying oven to dry, and obtain a deep eutectic solvent;

[0028] Step (4): Dissolve the biomacromolecule drug in distilled water to obtain an aqueous solution of the biomacromolecule drug. Add the aqueous solution of the biomacromolecule drug dropwise to the eutectic solvent and mix evenly to obtain the eutectic solvent-macromolecule drug delivery system.

[0029] Step (5): Prepare drug-loaded composite particles or drug-loaded composite microspheres by combining the deep eutectic solvent-macromolecule drug delivery system with excipients; encapsulate the drug-loaded composite particles or drug-loaded composite microspheres with an enteric protective layer to prepare enteric capsules, enteric tablets, enteric granules or enteric microspheres.

[0030] In step (1), preferably, the temperature of the water bath is 40°C. Generally, the reaction time is 12 hours.

[0031] In step (2), the temperature of the vacuum evaporation is 50℃~60℃, and the time of the vacuum evaporation is 2 hours.

[0032] In step (3), the drying temperature is 50℃~60℃ and the drying time is 24~48 hours.

[0033] In step (4), the concentration of the aqueous solution of the biological macromolecular drug is 1 to 100 mg / mL.

[0034] The volume ratio of the aqueous solution of the biomacromolecule drug to the deep eutectic solvent is 1:20 to 1:0.5, preferably 1:10 to 3:2, and more preferably 1:1.5 to 1:1.

[0035] Specifically, when the biopharmaceutical is low molecular weight heparin, the volume ratio of the aqueous solution of the biopharmaceutical to the eutectic solvent is 1:1.5 to 1:1; when the biopharmaceutical is calcitonin, the volume ratio of the aqueous solution of the biopharmaceutical to the eutectic solvent is 1:10 to 1:1; and when the biopharmaceutical is uricase, the volume ratio of the aqueous solution of the biopharmaceutical to the eutectic solvent is 1:1 to 3:2.

[0036] In step (5), when the dosage form of the macromolecular drug composition is enteric-coated capsules, the deep eutectic solvent-macromolecular drug is mixed with an appropriate amount of filler and wet granulation is performed to prepare drug-loaded composite particles; the drug-loaded composite particles are encapsulated into enteric-coated capsules to obtain enteric-coated capsules.

[0037] The ratio of the deep eutectic solvent to the filler is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg.

[0038] The enteric-coated capsules mentioned above are available in strengths of 20–25 mg.

[0039] When the dosage form of the macromolecular drug composition is an enteric-coated tablet, the deep eutectic solvent-macromolecular drug is mixed with an appropriate amount of filler and disintegrant, and wet granulation is performed to prepare drug-loaded composite particles; then, it is mixed with a lubricant and compressed into tablets using a tablet press to obtain tablet cores; the components of the enteric coating are dissolved in ethanol in sequence to prepare a coating solution; the tablet cores are coated using a coating machine and dried for 15-20 minutes to form an enteric coating layer, thus preparing enteric-coated tablets of macromolecular drugs.

[0040] The ratio of the total amount of the deep eutectic solvent to the filler and disintegrant is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg; the mass ratio of the filler to the disintegrant is 8:1 to 10:1, preferably 9:1.

[0041] The lubricant is 0.1% to 1% of the total mass of the biopharmaceutical, filler, and disintegrant.

[0042] The coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol at a mass ratio of (6.5-7.5):(6.5-7.5):(3.5-4.5):(0.8-1.2):1. The concentration of polyacrylic acid resin II in the coating solution is 1-10 mg / mL, preferably 1 mg / mL.

[0043] Preferably, the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol in a mass ratio of 7:7:4:1:1.

[0044] The mass of the enteric coating is 8% of the core mass.

[0045] The enteric-coated tablets are available in strengths of 20–25 mg.

[0046] When the dosage form of the macromolecular drug composition is enteric-coated granules, the deep eutectic solvent-macromolecular drug and an appropriate amount of filler are mixed and wet granulated to prepare drug-loaded composite granules; the components of the enteric coating are dissolved in ethanol in sequence to prepare a coating solution; the drug-loaded composite granules are coated using a coating machine and dried to form an enteric coating layer to prepare macromolecular drug enteric-coated granules.

[0047] The ratio of the deep eutectic solvent to the filler is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg.

[0048] The coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol at a mass ratio of 6.5-7.5:6.5-7.5:3.5-4.5:0.8-1.2:1. The concentration of polyacrylic acid resin II in the coating solution is 1-10 mg / mL, preferably 1 mg / mL.

[0049] Preferably, the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol in a mass ratio of 7:7:4:1:1.

[0050] When the dosage form of the macromolecular drug composition is enteric-coated microcapsules, the deep eutectic solvent-macromolecular drug and an appropriate amount of filler are mixed, extruded into thin strips by an extruder, granulated and sphericalized by a spheronizing machine, and then dried to obtain drug-loaded composite microcapsules; the components of the enteric coating are dissolved in ethanol in sequence to prepare a coating solution; the drug-loaded composite microcapsules are coated using a coating machine, dried for 15-20 minutes to form an enteric coating layer, and the macromolecular drug enteric-coated microcapsules are prepared.

[0051] The ratio of the deep eutectic solvent to the filler is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg.

[0052] The coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol at a mass ratio of (6.5-7.5):(6.5-7.5):(3.5-4.5):(0.8-1.2):1. The concentration of polyacrylic acid resin II in the coating solution is 1-10 mg / mL, preferably 1 mg / mL.

[0053] Preferably, the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol in a mass ratio of 7:7:4:1:1.

[0054] The mass of the enteric coating is 30% of the core mass.

[0055] The enteric-coated microcapsules or granules are available in doses of 20–25 mg.

[0056] The beneficial effects of this invention are:

[0057] The oral formulation of macromolecular drugs of the present invention has the advantages of simple preparation process, high safety, high stability and high oral bioavailability, and is also universally applicable to the development of a variety of oral formulations of macromolecular drugs.

[0058] This invention relates to oral formulations of macromolecular drugs based on deep eutectic solvent (DES) technology, which enhances the intestinal permeability of macromolecular drugs, thereby improving the oral bioavailability of low molecular weight heparin, calcitonin, and uricase.

[0059] This invention prepares DES-macromolecule drugs (i.e., deep eutectic solvent-macromolecule drug delivery system) into drug-loaded composite particles or drug-loaded composite microparticles, and then encapsulates them with enteric-coated capsules or enteric coatings, which can protect the drug from degradation by gastric acid and pepsin, reduce the first-pass effect in the digestive tract, and deliver it to the small intestine for targeted absorption.

[0060] Low molecular weight heparin oral formulations based on deep eutectic solvent technology, when the dosage of DES-LMWH oral enteric-coated capsules is 20 times that of the effective injection dose for preventing venous thromboembolism, can achieve the same effective blood concentration as the injection dose, and can be used for the prevention of venous thromboembolism. Calcitonin oral formulations based on deep eutectic solvent technology can significantly reduce blood calcium levels and can be used to treat osteoporosis and hypercalcemia from various causes. Uric acid oxidase oral formulations based on deep eutectic solvent technology can effectively improve the oral bioavailability of uricase and enhance drug stability, representing a potential technology for the oral delivery of uricase for the treatment of gout.

[0061] Compared with other permeation-enhancing methods, the oral formulation of this invention has a simple preparation process, low preparation cost, and does not involve organic solvents, conforming to the concept of "green chemistry" and applicable to the oral delivery of various macromolecular drugs. Existing research results show that choline or geranium acid alone cannot promote the intestinal permeability of drugs. However, the DES prepared by this invention using choline and geranium acid can protect macromolecular drugs from degradation by intestinal digestive enzymes and interact with the mucus layer, causing the mucus layer to thin, thereby promoting the paracellular transport of drugs. In addition, the raw materials used in DES are derived from natural products and are listed as "safe ingredients" by the U.S. Food and Drug Administration. While reversibly opening intercellular tight junctions and improving the efficiency of paracellular drug transport, it also has high biocompatibility. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the preparation process of the macromolecular drug composition based on deep eutectic solvent technology of the present invention.

[0063] Figure 2 The proton NMR spectrum of the deep eutectic solvent of this invention; wherein, Figure 2 A is the 1H NMR spectrum of choline, geraniol and DES 1:2; Figure 2 B represents the 1H NMR spectra of DES 3:2, DES 1:1, DES 1:2, and DES 1:4.

[0064] Figure 3 The Fourier transform infrared spectrum of the deep eutectic solvent of this invention; wherein, Figure 3 A represents the Fourier transform infrared spectrum of choline, geraniol, and DES in a 1:2 ratio; Figure 3 B represents the Fourier transform infrared spectra of DES 3:2, DES 1:1, DES 1:2, and DES 1:4.

[0065] Figure 4 Fourier transform infrared spectra of low molecular weight heparin, eutectic solvent, and oral formulations of eutectic solvent-low molecular weight heparin.

[0066] Figure 5 This is a dissolution curve of the low molecular weight heparin oral capsule of the present invention.

[0067] Figure 6 This is a graph showing the stability test results of the oral low molecular weight heparin formulation of this invention.

[0068] Figure 7 This is a diagram showing the in vitro release results of the oral low molecular weight heparin formulation of the present invention.

[0069] Figure 8 This is a blood concentration-time curve of the oral low molecular weight heparin formulation of the present invention in rats.

[0070] Figure 9 This is a blood concentration-time curve of the low molecular weight heparin oral capsules of the present invention in rats.

[0071] Figure 10 This is a section of intestinal tissue from rats after one week of continuous oral administration of low molecular weight heparin capsules.

[0072] Figure 11 This is a diagram showing the in vitro release results of the oral calcitonin formulation of this invention.

[0073] Figure 12 This is a blood concentration-time curve of the oral calcitonin formulation of the present invention in rats.

[0074] Figure 13 This is a graph showing the change in serum calcium ion levels in rats using the oral calcitonin formulation of this invention.

[0075] Figure 14 This is a graph showing the stability test results of the oral urate oxidase formulation of the present invention.

[0076] Figure 15 This is a diagram showing the in vitro release results of the oral urate oxidase formulation of the present invention.

[0077] Figure 16 This is a blood concentration-time curve of the oral urate oxidase preparation of the present invention in rats. Detailed Implementation

[0078] The present invention will be further illustrated below with reference to specific implementation examples, but the following embodiments do not limit the scope of protection of the present invention.

[0079] All raw materials or reagents used in this invention are commercially available.

[0080] The oral capsule formulations of macromolecular drugs were characterized using techniques such as nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and ultraviolet spectrophotometry.

[0081] We used instruments and methods such as peristaltic pumps, micro-chromogenic substrate methods, and ELISA kits to evaluate the intestinal permeability and oral bioavailability of macromolecular drugs in oral capsule formulations.

[0082] DES: Deep eutectic solvent; LMWH: Low molecular weight heparin; CT: Calcitonin; UOX: Uric acid oxidase; FITC-Dextran 4: fluorescein isothiocyanate-dextran; FITC-CT: fluorescein isothiocyanate-calcitonin; FITC-UOX: fluorescein isothiocyanate-uric acid oxidase.

[0083] Example 1: Synthesis and Characterization of DES

[0084] According to the molar ratio of choline to geranilic acid in Table 1, take an 80% (w / w) aqueous solution of choline bicarbonate and an 85% (w / w) aqueous solution of geranilic acid. Place an appropriate amount of the 80% (w / w) aqueous solution of choline bicarbonate in a 100 mL round-bottom flask, heat in a 40°C water bath, and add an appropriate amount of the 85% (w / w) aqueous solution of geranilic acid dropwise to the choline bicarbonate aqueous solution under stirring until no carbon dioxide is released (no bubbles are visually observed). Transfer the round-bottom flask to a rotary evaporator and evaporate at 60°C for 2 hours to remove the solvent. Place the product in a vacuum drying oven and dry at 60°C for 24 hours to prepare a series of DES.

[0085] Nuclear magnetic resonance (NMR) spectroscopy and infrared spectroscopy were used to determine the proton NMR and infrared spectra of choline, geraniol, and DES, respectively. The successful synthesis of DES was verified by analyzing the chemical shifts of hydrogen atoms in the proton NMR spectrum and the peak shifts of characteristic peaks in the infrared spectrum.

[0086] Table 1: Different molar ratios of choline and geranilic acid in synthesis and the preparation of DES

[0087]

[0088] like Figure 2 As shown in Figure A, the 1H NMR spectrum of DES exhibits characteristic peaks for choline and geranilic acid. Due to the hydrogen bond between choline and geranilic acid, the hydroxyl peak of choline disappears from the 1H NMR spectrum of DES, indicating the successful synthesis of DES. Furthermore, Figure 2 B shows that in the 1H NMR spectra of DES prepared with different molar ratios of choline and geranilic acid, the methyl hydrogens in both choline and geranilic acid underwent certain shifts. As the ratio of choline to geranilic acid increased, the hydrogen atoms in the DES shifted to lower fields.

[0089] like Figure 3 As shown in Figures A and 3B, the infrared characteristic peaks of the hydroxyl group in choline and the carbonyl group in geraniol both exhibit a certain degree of shift in their infrared spectra, indicating that DES was successfully synthesized.

[0090] Example 2: Preparation and Characterization of Oral Formulation of LMWH

[0091] Multiple LMWH solutions were prepared. The preparation method for each LMWH solution was as follows: 5 mg of low molecular weight heparin (LMWH, enoxaparin sodium was used in this example) was dissolved in 1 mL of distilled water and mixed well to obtain an LMWH solution. The above LMWH solution was added dropwise to 1 mL of DES (DES1:4, DES1:2, DES1:1, and DES3:2, respectively) and vortexed to obtain DES-LMWH (eutectic solvent-low molecular weight heparin, the DES-LMWH corresponding to DES1:4, DES1:2, DES1:1, and DES3:2 are respectively denoted as DES(1:4)-LMWH, DES(1:2)-LMWH, DES(1:1)-LMWH, and DES(3:2)-LMWH), and the drug concentration of LMWH was 2.5 mg / mL.

[0092] The DES1:2, LMWH, and DES(1:2)-LMWH samples were dried, and their infrared spectra were measured using Fourier transform infrared spectroscopy. The results are as follows: Figure 4 As shown, after LMWH dissolves in DES (1:2), no new covalent bonds are formed between the two. The characteristic peaks of LMWH are not displayed in DES (1:2)-LMWH, indicating that LMWH is completely dissolved in DES and that DES (1:2)-LMWH is an organic whole.

[0093] Example 3: Screening of the preparation process for DES(1:2)-LMWH enteric-coated capsules

[0094] Examining the types of fillers: Following the formulations in Table 2, lactose, mannitol, or sorbitol were used as fillers. DES(1:2)-LMWH (also used as a binder) was mixed with a certain amount of filler and granulated according to the standard of "forming a clump when squeezed but crumbling upon contact." The granules were then sieved through a 20-mesh sieve and dried in a 60℃ constant temperature drying oven for 3 hours to obtain drug-loaded composite particles. These particles were then encapsulated in enteric-coated capsules (enteric-coated capsules for rats, Shanghai Yuyan Scientific Instruments Co., Ltd.) to obtain DES(1:2)-LMWH enteric-coated capsules. By comparing the drug content and content uniformity of the enteric-coated capsules obtained from each formulation, a more suitable filler was selected.

[0095] Table 2: Formulation composition for consideration of filler types

[0096]

[0097] Table 3: Drug content and content uniformity (%) of formulations with different filler compositions

[0098]

[0099] Note: A is the absolute value of the difference between the indicated quantity and the mean, and S is the standard deviation.

[0100] As shown in Table 3, the content uniformity of capsules made with mannitol and lactose as fillers was 7.36% and 10.93%, respectively, both less than 15%, which meets the pharmacopoeia requirements. However, sorbitol, due to its strong hygroscopicity, resulted in a soft mass with too much viscosity after sieving, failing to form granules. Furthermore, ideal pharmaceutical excipients should have good safety profiles. Previous studies have shown that mannitol has a higher safety profile compared to lactose and sorbitol. Lactose intolerance and fructose intolerance are common adverse reactions to excipients; approximately two-thirds of the global population suffers from varying degrees of lactose intolerance, and about 30% of Westerners and 10% of Asians suffer from fructose intolerance. Considering both drug content uniformity and excipient safety, mannitol was chosen as the filler for enteric-coated capsules.

[0101] Investigation of binder dosage: According to the prescription in Table 4, different amounts of DES (1:2) were used as binders and mixed with a certain amount of mannitol. Enteric-coated capsules were prepared according to the method of "investigation of filler type". The drug content and content uniformity of the capsules obtained from each prescription were compared to determine the appropriate amount of binder.

[0102] Table 4: Formulation composition for adhesive dosage assessment

[0103]

[0104] Table 5: Drug content and content uniformity (%) of formulations with different amounts of adhesive.

[0105]

[0106] As shown in Table 5, when the amount of DES (1:2) used per 500 mg of mannitol was 250, 300, and 350 μL, the content uniformity of the resulting capsules was 10.98%, 7.36%, and 12.60%, respectively, all less than 15%, which meets the pharmacopoeia requirements. Among these, the content uniformity of the capsules was lowest when the amount of DES (1:2) was 300 μL, indicating a more uniform distribution of the drug within the capsules. Therefore, 300 μL was selected as the optimal amount of DES (1:2) binder.

[0107] The optimal preparation process for DES(1:2)-LMWH enteric-coated capsules is as follows: Dissolve 20 mg LMWH in 300 μL of distilled water, add it dropwise to 300 μL of DES(1:2), and vortex mix to obtain DES(1:2)-LMWH. DES(1:2)-LMWH also serves as a binder, and is mixed with 500 mg of mannitol. The mixture is prepared into a soft mass according to the standard of "forming a clump when squeezed, but crumbling upon contact," and granulated through a 20-mesh sieve. The granules are dried in a constant temperature drying oven at 60℃ for 3 hours to obtain drug-loaded composite particles. These particles are then encapsulated in enteric-coated capsules (enteric-coated capsules for rats, Shanghai Yuyan Scientific Instruments Co., Ltd.) to obtain DES(1:2)-LMWH enteric-coated capsules. The oral enteric-coated capsule specification is 25 mg.

[0108] Example 4: Preparation of DES(1:2)-LMWH enteric-coated tablets, enteric-coated granules and enteric-coated microcapsules

[0109] Based on the screening results of Example 3, DES(1:2)-LMWH enteric-coated tablets, enteric-coated granules and enteric-coated microcapsules were prepared using mannitol as a filler and the ratio of LMWH to DES(1:2) was 20:300 mg / μL.

[0110] Preparation of DES(1:2)-LMWH enteric-coated tablets: 20 mg LMWH was dissolved in 300 μL of distilled water and added dropwise to 300 μL of DES(1:2). The mixture was vortexed and mixed to obtain DES(1:2)-LMWH. DES(1:2)-LMWH also served as a binder. It was mixed with 450 mg mannitol and 50 mg dry starch, granulated by sieving through a 20-mesh sieve, and dried in a constant temperature drying oven at 60 °C for 3 h to obtain drug-loaded composite granules. Then, it was mixed with 2.5 mg magnesium stearate (magnesium stearate was 0.48% of the total mass of LMWH + mannitol + dry starch after drying) and compressed into tablets using a tablet press to obtain tablet cores. 14 mg of polyacrylic acid resin II, 14 mg of polyacrylate III, 8 mg of castor oil, 2 mg of polysorbate 80, and 2 mg of dimethyl phthalate were dissolved in anhydrous ethanol to prepare a coating solution (the concentration of polyacrylic acid resin II was 1 mg / mL). The tablet core was coated using a coating machine, and the mass of the enteric coating layer was 8% of the mass of the tablet core. After coating, the tablets were dried for 20 min to prepare DES-LMWH oral enteric-coated tablets. The oral enteric-coated tablets were in the specification of 25 mg.

[0111] Preparation of DES(1:2)-LMWH enteric-coated granules: 20 mg LMWH was dissolved in 300 μL of distilled water and added dropwise to 300 μL of DES(1:2). The mixture was vortexed and mixed to obtain DES(1:2)-LMWH. DES(1:2)-LMWH also served as a binder and was mixed with 500 mg of mannitol. The mixture was prepared into a soft mass according to the standard of "forming a clump when squeezed but dispersing when touched". The prepared soft mass was granulated by passing it through a 20-mesh sieve and dried in a constant temperature drying oven at 60℃ for 3 h to obtain drug-loaded composite granules. 52.5 mg of polyacrylic acid resin II, 52.5 mg of polyacrylate III, 30 mg of castor oil, 7.5 mg of polysorbate 80, and 7.5 mg of dimethyl phthalate were dissolved in ethanol in sequence to prepare a coating solution (the concentration of polyacrylic acid resin II was 1 mg / mL). The drug-loaded composite particles were coated using a coating machine, and the mass of the enteric coating was 30% of the mass of the pellet core. After coating, the pellets were dried for 20 min to prepare DES(1:2)-LMWH oral enteric-coated granules. The oral enteric-coated granules were in a specification of 25 mg.

[0112] Preparation of DES(1:2)-LMWH enteric-coated microcapsules: 20 mg LMWH was dissolved in 300 μL of distilled water and added dropwise to 300 μL of DES(1:2). The mixture was vortexed and mixed to obtain DES(1:2)-LMWH. DES(1:2)-LMWH also served as a binder and was mixed with 500 mg of mannitol. The mixture was prepared into a soft material according to the standard of "forming a ball when squeezed but dispersing when touched". It was extruded into thin strips through an extruder and quickly fed into a spheroidizer for granulation and spheroidization. Finally, it was dried in a constant temperature drying oven at 60 °C for 3 h to prepare drug-loaded composite microcapsules. 52.5 mg of polyacrylic acid resin II, 52.5 mg of polyacrylate III, 30 mg of castor oil, 7.5 mg of polysorbate 80, and 7.5 mg of dimethyl phthalate were dissolved in ethanol in sequence to prepare a coating solution (the concentration of polyacrylic acid resin II was 1 mg / mL). The drug-loaded composite microcapsules were coated using a coating machine, and the mass of the enteric coating layer was 30% of the mass of the core. After coating, the microcapsules were dried for 20 min to prepare DES(1:2)-LMWH oral enteric microcapsules with a specification of 25 mg.

[0113] Example 5: Quality evaluation of LMWH oral formulation

[0114] In accordance with the requirements of granules in General Chapter 0104 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the particle size, loss on drying and solubility of the drug-loaded composite particles in the DES(1:2)-LMWH enteric-coated capsules in Example 3, the drug-loaded composite particles in the DES(1:2)-LMWH enteric-coated tablets in Example 4, and the drug-loaded composite microspheres in the DES(1:2)-LMWH enteric-coated microspheres were investigated.

[0115] Table 6: Quality evaluation results of the low molecular weight heparin-loaded composite particles of the present invention

[0116]

[0117] As shown in Table 6, the total amount of particles passing through sieve No. 1 and sieve No. 5 is less than 15%, the loss on drying is less than 2%, and the particles can be completely dissolved in hot water within 5 minutes, which meets the requirements of the pharmacopoeia.

[0118] In accordance with the requirements for capsules in General Chapter 0103 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the appearance, water content, fill weight variation, content uniformity and dissolution of the DES(1:2)-LMWH enteric-coated capsules in Example 3 were examined to determine whether the capsules met the quality standards stipulated in the Pharmacopoeia.

[0119] The specific steps for dissolution testing were as follows: Using the basket method from the pharmacopoeia, the dissolution of DES(1:2)-LMWH enteric-coated capsules in simulated gastric fluid (SGF, containing 84 mmol / L HCl and 34 mmol / L NaCl) and simulated intestinal fluid (SIF, containing 50 mmol / L KH2PO4 and 24 mmol / L NaOH) were investigated. 300 mL of SGF and 300 mL of SIF were measured and placed in dissolution vessels. Once the temperature of the dissolution medium was constant at 37 ± 0.5 °C, the DES(1:2)-LMWH enteric-coated capsules were added to the dissolution vessels at a rotation speed of 100 r / min. First, the dissolution in SGF was investigated over 2 hours. At 5, 10, 15, 30, 45, 60, 90, and 120 minutes, 6 mL of the dissolved solution was taken from the dissolution vessel and replenished with an equal volume of fresh SGF. Next, the dissolution medium was changed, and the dissolution amount in SIF was examined over 4 hours. At mins of 5, 10, 15, 30, 45, 60, 90, 120, and 240, 6 mL of dissolution solution was taken from the dissolution vessel and replenished with an equal volume of fresh SIF. The solution was filtered through a 0.45 μm aqueous filter membrane, and the absorbance of LMWH was measured using ultraviolet spectrophotometry. The cumulative dissolution amount of DES(1:2)-LMWH enteric-coated capsules in different media was calculated.

[0120] Table 7: Quality Evaluation Results of the Low Molecular Weight Heparin Enteric-Coated Capsules of the Present Invention

[0121]

[0122] As shown in Table 7, the DES(1:2)-LMWH enteric-coated capsules have a clean and smooth appearance and no odor. The drug content in the capsules is 98.51%, the water content is 3.06%, the fill weight variation is 1.63%, and the content uniformity is 7.36%, all of which meet the pharmacopoeia standards. Figure 5As shown, DES(1:2)-LMWH enteric-coated capsules showed almost no dissolution in artificial gastric fluid, and the cumulative dissolution in artificial intestinal fluid was over 85%, which meets the pharmacopoeia requirements.

[0123] Example 6: Intestinal permeability study of DES-LMWH

[0124] Fifteen male SD rats (weighing approximately 250–300 g) were randomly divided into five groups according to their body weight: Free-LMWH group, DES(1:4)-LMWH group, DES(1:2)-LMWH group, DES(1:1)-LMWH group, and DES(3:2)-LMWH group.

[0125] FITC-Dextran 4 is a FITC-labeled dextran with an average molecular weight of approximately 4000 Da, and it has a similar chemical composition and physicochemical properties to LMWH. To simplify the detection method and visualize the experimental phenomenon, FITC-Dextran 4 was selected as an alternative drug to LMWH for in vivo one-way intestinal perfusion experiments in rats.

[0126] 1 mg of FITC-Dextran 4 was dissolved in water to prepare a FITC-Dextran 4 solution (Dextran 4 concentration: 2 mg / mL, FITC concentration: 1 mg / mL). This solution was diluted with Krebs-Ringer buffer and DES (DES 3:2, DES 1:1, DES 1:2, DES 1:4, etc.) was added, followed by vortexing to prepare a DES-FITC-Dextran 4 solution (Dextran 4 concentration: 0.05 mg / mL, DES volume percentage: 25%). Rats in the Free-LMWH group were given the FITC-Dextran 4 solution, while rats in each DES-LMWH group were given DES-FITC-Dextran 4 solutions containing different amounts of DES. Rats were fasted for 24 hours prior to the experiment. The intestinal contents were first flushed with physiological saline, then the intestinal segments were equilibrated with Krebs-Ringer buffer at pH 7.4 at a flow rate of 0.5 mL / min for equilibration over a period of 0.5 h. After equilibration, the perfusion fluid was replaced with the respective formulations, and the perfusion rate was 0.2 mL / min for 2 h. After the experiment, the length and inner radius of the small intestine were dissected and measured. The volume of the perfusion fluid was corrected using a gravimetric method, and the drug concentrations before and after perfusion were determined using a micro-biocolor assay to calculate the effective intestinal permeability coefficient P for each formulation. eff and absorption rate constant K a Assess its intestinal permeability and optimize the formulation.

[0127] Calculation formula:

[0128] Peff =[-Q*In(C out ·V out / C in ·V in )] / 2*π*l*r

[0129] K a =Q*(1-C out ·V out / C in ·V in ) / (π*r*r*l)

[0130] C in and C out These represent the mass concentrations (mg / mL) of the intestinal inlet and outlet perfusion fluids, respectively; V in and V out , respectively, represent the corrected intestinal inlet and outlet perfusion volumes (mL); l and r represent the length (cm) and cross-sectional radius (cm) of the perfused intestinal segment, respectively; Q represents the perfusion rate (mL / min).

[0131] Table 8: Results of intestinal permeability test of the oral low molecular weight heparin formulation of the present invention

[0132]

[0133] Note: Compared with the Free-LMWH group, *P<0.05, **P<0.01, ***P<0.001.

[0134] As shown in Table 8, DES with different molar ratios of choline and geraniol all improved the intestinal permeability of FITC-Dextran 4, and the effect was influenced by the proportion of the DES components. (Regarding P...) eff One-way ANOVA revealed that only the group with a choline to geraniol molar ratio of 1:2 (DES1:2) showed a significant difference from the other groups; K a One-way ANOVA revealed statistically significant differences among the groups, with the DES(1:2)-LMWH group showing the most significant difference from the other groups. Therefore, DES1:2 was selected as the optimal DES for preparing DES-LMWH.

[0135] Example 7: Stability Study of DES(1:2)-LMWH

[0136] To investigate the stability of DES(1:2)-LMWH, the drug content in DES(1:2)-LMWH was determined by ultraviolet spectrophotometry. The specific steps were as follows: 0.5 mg LMWH was accurately weighed and added to 0.5 mL of distilled water, mixed well to obtain an LMWH aqueous solution; the LMWH aqueous solution was added to 0.5 mL of DES(1:2), vortexed to prepare a DES(1:2)-LMWH solution with a concentration of 0.5 mg / mL, and stored at (4±1)℃ and (25±3)℃, respectively. On days 0, 1, 7, and 14, 20 μL of sample solution was taken into a 10 mL volumetric flask, 0.5 mL of 0.05 mg / mL methylene blue was added, and the solution was diluted to the mark with water. After reacting at room temperature for 1 h, the absorbance value was measured, and the drug concentration was calculated to assess the drug stability.

[0137] The results are as follows Figure 6 As shown, the drug content in the formulation remained stable under both temperature conditions. The significance level between the groups was P = 0.643 > 0.05, indicating that there was no significant difference in the stability of the formulation at 4℃ and 25℃.

[0138] Example 8: In vitro release study of DES(1:2)-LMWH

[0139] To investigate the release of DES(1:2)-LMWH in vivo, an in vitro simulation method was used to determine the cumulative release of LMWH under different pH conditions. The specific steps were as follows: 50 mg of LMWH was accurately weighed and added to 2.5 mL of distilled water, mixed thoroughly to obtain an LMWH aqueous solution; the LMWH aqueous solution was added to 2.5 mL of DES(1:2), vortexed, and mixed to prepare a DES(1:2)-LMWH concentration of 10 mg / mL. The in vitro release experiment was conducted in a constant temperature water bath shaker (37℃, 100 rpm). 4.5 mL of DES(1:2)-LMWH was divided into nine equal portions and placed in dialysis bags (MWCO: 8000 Da), and then placed in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH 6.8), and phosphate buffer (pH 7.4), respectively, with three replicates for each medium. Samples of 200 μL were taken at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 hours after the start of the experiment, and an equal volume of fresh release medium was added simultaneously. The concentration of LMWH at each time point was determined by ultraviolet spectrophotometry, and the cumulative release of LMWH was calculated.

[0140] The results are as follows Figure 7As shown, the release rate of DES-LMWH was zero at pH 1.2. This is likely because LMWH is destroyed in an acidic environment, resulting in extremely low drug concentrations in the sample. This also suggests that the formulation may be destroyed in the gastric environment, requiring bypassing the gastric environment or enteric coating during administration. After 36 hours of sustained release of DES(1:2)-LMWH in phosphate buffer at pH 6.8 and pH 7.4, the drug concentrations inside and outside the dialysis bag tended to reach equilibrium. The cumulative release of DES(1:2)-LMWH was highest at pH 7.4, approximately 60%.

[0141] Example 9: Oral bioavailability study of DES(1:2)-LMWH

[0142] The preparation of DES(1:2)-LMWH is the same as in Example 2.

[0143] Oral administration is simulated via intestinal injection. Intestinal injection: A segment of the rat's small intestine is surgically removed, the drug is dispersed in water, and administered via injection.

[0144] Fifteen male SD rats (weighing approximately 250±20g) were randomly divided into three groups according to body weight: Free LMWH group, low-dose DES(1:2)-LMWH group, and high-dose DES(1:2)-LMWH group. Rats in the high-dose and low-dose DES(1:2)-LMWH groups were administered DES(1:2)-LMWH 40mg / kg and 20mg / kg respectively via enteral injection, while rats in the Free LMWH group were administered LMWH aqueous solution (5mg / kg) subcutaneously. Rats were fasted for 24 hours prior to the experiment. Blood samples (0.5mL / sample) were collected from the medial canthal venous plexus of the rats at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 hours of the experiment. The anti-FXa activity of LMWH in plasma samples was determined by micro-chromogenic substrate method, and the plasma concentration of the drug was calculated to evaluate the oral bioavailability of DES(1:2)-LMWH.

[0145] According to relevant literature, a subcutaneous injection dose of 5 mg / kg of LWMH resulted in an effective therapeutic concentration in rats. The plasma concentration-time curve and pharmacokinetic parameters of LWMH in rats are shown below. Figure 8 As shown in Table 9, when the oral dose of the DES(1:2)-LMWH formulation is 4 times the effective therapeutic dose for injection, the effective blood concentration for injection can be achieved.

[0146] Table 9: Pharmacokinetic parameters of the oral low molecular weight heparin formulation of the present invention in rats.

[0147]

[0148] Example 10: Bioavailability Study of DES(1:2)-LMWH Enteric-coated Capsules

[0149] Preparation of LMWH+mannitol oral enteric-coated capsules: 20mg LMWH and 500mg mannitol were mixed, and an appropriate amount of water was added. The mixture was prepared into a soft mass according to the standard of "forming a clump when squeezed but crumbling when touched". The granules were sieved through a 20-mesh sieve and dried in a constant temperature drying oven at 60℃ for 3 hours. The granules were then encapsulated in enteric-coated capsules (enteric-coated capsules for rats, Shanghai Yuyan Scientific Instruments Co., Ltd.) to obtain LMWH+mannitol oral enteric-coated capsules. The specification of the oral enteric-coated capsules is 25mg.

[0150] The preparation of DES(1:2)-LMWH enteric-coated capsules is the same as in Example 3.

[0151] Twenty male SD rats (weighing approximately 250±20g) were randomly divided into four groups according to their body weight: a subcutaneous injection Free LMWH group, a Free LMWH control group, an LMWH+mannitol oral enteric-coated capsule control group, and a DES(1:2)-LMWH oral enteric-coated capsule group. The Free LMWH group received a subcutaneous injection of LMWH aqueous solution (1mg / kg), the Free LMWH control group received LMWH-only enteric-coated capsules (20mg / kg) by gavage, the LMWH+mannitol oral capsule control group received LMWH and mannitol-containing enteric-coated capsules (20mg / kg) by gavage, and the DES(1:2)-LMWH oral capsule group received DES(1:2)-LMWH enteric-coated capsules (20mg / kg). The rats were fasted for 24 hours before the experiment. Blood samples (0.5 mL / sample) were collected from the medial canthal venous plexus of rats at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 hours of the experiment. The anti-FXa activity of LMWH in the plasma samples was determined by microchromogenic substrate method, and the plasma concentration of the drug was calculated to assess the oral bioavailability of the formulation.

[0152] According to relevant literature, a subcutaneous injection dose of 1 mg / kg of LWMH in rats achieves an effective preventative blood concentration. The blood concentration-time curve and pharmacokinetic parameters of LWMH in rats are shown below. Figure 9 As shown in Table 10, when DES(1:2)-LMWH enteric-coated capsules (20 mg / kg) were administered by gavage, C max The concentration was 0.56 IU / mL, and the AUC was... 0-24hThe effective blood concentration was 2.75 IU·h / mL, reaching the level required for injection, and the duration of effective prevention of blood concentration was at least 8 hours. The absolute bioavailability of DES(1:2)-LMWH enteric-coated capsules was 19.93%, and the relative bioavailability was 189.66%, which were significantly higher than those of the free LMWH control group and the LMWH+mannitol oral enteric-coated capsule control group, indicating that the oral bioavailability of LMWH can be significantly improved by making DES(1:2)-LMWH into enteric-coated capsules.

[0153] Table 10: Pharmacokinetic parameters of the low molecular weight heparin oral capsules of the present invention in rats.

[0154]

[0155] Example 11: Safety evaluation of DES(1:2)-LMWH enteric-coated capsules

[0156] The preparation of DES(1:2)-LMWH enteric-coated capsules is the same as in Example 3.

[0157] Six male SD rats (approximately 250±20g) were randomly divided into two groups according to body weight: a control group and a formulation group. The control group was orally administered 0.9% saline (1mL / rat), while the formulation group was administered DES(1:2)-LMWH enteric-coated capsules (20mg / kg). Administration was once daily for 7 consecutive days. On the eighth day after administration, the rats were sacrificed, and their duodenum, jejunum, and ileum were removed and fixed with 4% paraformaldehyde. The fixed intestinal segments were prepared into paraffin sections and stained with H&E. The morphology of the small intestine was observed under an upright fluorescence microscope to determine whether there were significant changes in the tissue morphology of the DES(1:2)-LMWH oral enteric-coated capsule group, thereby assessing the irritant effect of the formulation on the intestine.

[0158] like Figure 10 As shown, the transverse section of the intestine of the control group rats was intact, with tightly arranged annular folds and clear boundaries; compared with the control group, there were no significant differences in the various intestinal segments of the formulation group rats. This indicates that after continuous administration of DES(1:2)-LMWH enteric-coated capsules for 7 days, DES(1:2) did not exhibit intestinal toxicity, and no obvious intestinal irritation was observed in the rats, indicating that the rats tolerated the oral enteric-coated capsules of DES(1:2)-LMWH well.

[0159] Example 12: Preparation of DES-CT and Study of Intestinal Permeability

[0160] The specific steps for preparing DES-CT are as follows: Dissolve 4 mg of calcitonin (CT) in 1 mL of distilled water, mix well, and add the above CT solution dropwise to 1 mL of DES (DES2:1, DES1:1, and DES1:2 respectively), vortex, and obtain DES-CT (DES(2:1)-CT, DES(1:1)-CT, and DES(1:2)-CT respectively), with a drug concentration of 2 mg / mL.

[0161] The intestinal permeability of DES-CT was studied by preparing DES-FITC-CT: 12 mg of CT was accurately weighed and dissolved in 0.1 M sodium carbonate buffer at pH 9.0 as the aqueous phase (final CT concentration 2 mg / mL); 0.3 mg of FITC was accurately weighed and dissolved in 0.3 mL of DMSO as the organic phase (final FITC concentration 1 mg / mL). Under ice bath conditions, an organic phase was added to the aqueous phase, and the mixture was magnetically stirred for 8 hours in the dark. 19 mg of ammonium chloride was accurately weighed and dissolved in 1 mL of water to prepare a reaction termination solution. The termination solution was added to the reaction solution, and the mixture was stirred for another 2 hours at 4°C in the dark. The resulting solution was added to an ultrafiltration tube (MWCO 3000 Da), centrifuged at 4000 rpm for 10 min to remove free FITC, and the supernatant was collected. The solution was diluted with Krebs-Ringer buffer to dilute the CT concentration to 2 mg / mL. DES (DES 2:1, DES 1:1, and DES 1:2, respectively) was then added and vortexed to prepare a DES-FITC-CT solution (CT concentration of 0.05 mg / mL and DES volume percentage of 10%).

[0162] The specific steps of the DES-CT intestinal permeability study were as follows: Twelve male SD rats (weighing approximately 250–300 g) were randomly divided into four groups according to their body weight: Free-CT group, DES(2:1)-CT group, DES(1:1)-CT group, and DES(1:2)-CT group. In vivo one-way intestinal perfusion was performed, using FITC-CT instead of the drug CT for small intestinal perfusion. The Free-CT group was given FITC-CT solution (prepared according to the aforementioned method, collecting the supernatant from the ultrafiltration tube and diluting it with Krebs-Ringer buffer to a CT concentration of 50 μg / mL). The three DES-CT groups were given DES-FITC-CT solutions prepared with different molar ratios of choline and geraniol, respectively. The specific steps of the in vivo one-way intestinal perfusion and the intestinal permeability assessment were the same as in Example 6.

[0163] As shown in Table 11, both DES-FITC-CT and FITC-CT can be absorbed by the jejunum. The absorption rate constant K of the DES(1:2)-CT group... aThe apparent absorption coefficient P of the DES(1:2)-CT group was approximately twice that of the Free-CT group. eff Approximately 1.6 times that of the Free-CT group, and the K value of the DES(1:2)-CT group. a With P eff There was a significant difference between the control group and the control group (P<0.05). This indicates that DES can promote jejunal absorption of CT, and DES1:2 has the best permeation-promoting effect. Therefore, DES1:2 was selected as the optimal DES in the DES-CT prescription.

[0164] Table 11: Results of intestinal permeability test of the oral calcitonin formulation of the present invention

[0165]

[0166] Note: *P<0.05 compared with the Free-CT group.

[0167] Example 13: In vitro release study of DES(1:2)-CT

[0168] To investigate the release of DES(1:2)-CT in vivo, the cumulative release of CT under different pH conditions was determined using an in vitro simulation method. The specific steps were as follows: An in vitro release experiment was conducted in a constant temperature water bath shaker (37℃, 100 rpm). Following Example 12, a DES(1:2)-FITC-CT solution (CT concentration of 0.05 mg / mL, DES volume percentage of 10%) was prepared. The solvent was removed, and 5.4 mL of DES(1:2)-FITC-CT solution (CT concentration of 2 mg / mL) was prepared with water. This 5.4 mL DES(1:2)-FITC-CT solution was divided into nine equal portions and placed in dialysis bags (MWCO: 8000 Da). These portions were then placed in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH 6.8), and phosphate buffer (pH 7.4), respectively, with three replicates for each medium. Samples of 1 mL were taken at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 hours after the start of the experiment, and an equal volume of fresh release medium was added simultaneously. The fluorescence intensity of CT at each time point was measured using a Cytation5 multimode microplate detection and cell imaging system at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the cumulative release was calculated.

[0169] like Figure 11As shown, DES-CT releases slowly in buffer solutions. At 10 hours, CT is completely released in pH 7.4 buffer; at 12 hours, 97% of CT is released in pH 6.8 buffer, with a slight decrease in release thereafter, possibly due to degradation. Only 8% of CT is released in pH 1.2 buffer. DES-CT remains stable in pH 7.4 buffer, and the release rate is faster in pH 7.4 buffer.

[0170] Example 14: Oral bioavailability study of DES(1:2)-CT

[0171] Twelve male SD rats (weighing approximately 250±20g) were randomly divided into three groups according to body weight: a Free CT subcutaneous injection group, a DES(1:2)-CT intestinal injection group, and a Free CT intestinal injection control group. The Free CT subcutaneous injection group received CT aqueous solution (0.045mg / kg), the DES(1:2)-CT intestinal injection group received DES(1:2)-CT (1.35mg / kg, prepared according to the method in Example 12, with CT concentration of 0.05mg / mL and DES volume percentage of 10%), the solvent was removed, and then dissolved in water before administration. The Free CT intestinal injection control group received CT aqueous solution (1.35mg / kg). Rats were fasted for 24 hours before the experiment. Blood samples (0.5mL / sample) were collected from the inner canthal venous plexus of the rats at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 hours of the experiment. The concentration of CT in the plasma of the sample was determined by an ELISA kit, and the pharmacokinetic parameters were calculated to evaluate the oral bioavailability of DES(1:2)-CT.

[0172] The plasma concentration-time curve of CT in rats is shown below. Figure 12 As shown in Table 12, the bioavailability of the oral DES-CT formulation is approximately 4.8 times that of the aqueous CT solution, with a relative bioavailability of 3.43%.

[0173] Table 12: Pharmacokinetic parameters of the oral calcitonin formulation of the present invention in rats.

[0174]

[0175] Example 15: Pharmacodynamic study of DES(1:2)-CT

[0176] Sixteen male SD rats (weighing approximately 250±20g) were randomly divided into four groups according to body weight: a subcutaneous injection group of Free CT, an intestinal injection group of DES (1:2)-CT, a positive control group, and a negative control group. The subcutaneous injection group received CT aqueous solution (0.045mg / kg), the intestinal injection group received DES (1:2)-CT (1.35mg / kg, prepared as in Example 12), the positive control group received intestinal injection of CT aqueous solution (1.35mg / kg), and the negative control group received an equal volume of physiological saline. All rats were fasted for 24 hours before the experiment. Blood samples (0.5mL / sample) were collected from the venous plexus at the inner canthus of the rats' eyes at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 24 hours. After centrifugation, the supernatant was separated, and the calcium ion concentration was determined using the o-cresolphthalein complexone colorimetric method.

[0177] like Figure 13 As shown, 8 hours after subcutaneous injection of CT aqueous solution, serum calcium levels rapidly decreased to below 60%, with a significant drop in serum calcium. The oral DES(1:2)-CT formulation also exhibited a certain hypocalcemic effect; after 8 hours of intestinal injection, serum calcium concentration decreased to 68% of the initial concentration, reaching its lowest value.

[0178] Example 16: Preparation and Intestinal Permeability Study of DES-UOX

[0179] The specific steps for preparing DES-UOX are as follows: Dissolve 5 mg of urate oxidase (UOX) in 1 mL of distilled water, mix well, add the above UOX solution dropwise to 1 mL of DES2:1, vortex, and obtain DES(2:1)-UOX with a drug concentration of 2.5 mg / mL.

[0180] The intestinal permeability of DES-UOX was studied by preparing DES-FITC-UOX: 30 mg of UOX was accurately weighed and dissolved in 0.1 M sodium carbonate buffer at pH 9.0 as the aqueous phase (final concentration 5 mg / mL). 1 mg of FITC was accurately weighed and dissolved in 1 mL of DMSO as the organic phase (final concentration 1 mg / mL). Under ice bath conditions, 0.9 mL of the organic phase was added to the aqueous phase, and the mixture was magnetically stirred for 8 h in the dark. 0.27 g of ammonium chloride was accurately weighed and dissolved in 1 mL of water to prepare the reaction termination solution. The stop solution was added dropwise to the reaction solution until the UOX concentration reached 50 mM, and the reaction was terminated for 2 h to obtain a yellow-green fluorescently labeled drug solution. The obtained solution was added to an ultrafiltration tube (MWCO 3000 Da), centrifuged at 4000 rpm for 10 min to remove free FITC, and the supernatant of the ultrafiltration tube was collected and diluted with Krebs-Ringer buffer to dilute the UOX concentration to 8 mg / mL. DES 2:1 was added and vortexed to prepare a DES-FITC-UOX solution (UOX concentration of 0.06 mg / mL, DES 2:1 volume percentage of 10%).

[0181] The specific steps for the DES(2:1)-UOX intestinal permeability study were as follows: Six male SD rats (weighing approximately 250–300 g) were randomly divided into two groups according to their body weight: the Free-UOX group and the DES(2:1)-UOX group. In vivo one-way intestinal perfusion was performed, using FITC-UOX instead of the drug UOX for small intestinal perfusion. The Free-UOX group was given FITC-UOX solution (prepared according to the aforementioned method, collecting the supernatant from the ultrafiltration tube and diluting it with Krebs-Ringer buffer to a UOX concentration of 0.06 mg / mL), while the DES(2:1)-UOX group was given DES(2:1)-FITC-UOX solution (UOX concentration of 0.06 mg / mL). The specific steps for the in vivo one-way intestinal perfusion and the intestinal permeability assessment were the same as in Example 6.

[0182] Table 13: Results of intestinal permeability test of the oral uricase preparation of the present invention

[0183]

[0184] Note: *P < 0.05 compared to the Free-UOX group.

[0185] As shown in Table 13, compared to the Free-UOX group, the absorption rate constant K of the DES(2:1)-UOX group is... a The ratio was significantly higher than that of the control group (P<0.05), indicating that DES2:1 also has a certain promoting effect on the apparent absorption coefficient of the drug. Therefore, DES2:1 was selected as the optimal DES in the DES-CT formulation.

[0186] Example 17: Stability Study of DES(2:1)-UOX

[0187] To investigate the stability of DES(2:1)-UOX, circular dichroism spectroscopy was used to detect the secondary conformational changes of uricase. Specifically, the DES(2:1)-UOX solution prepared in Example 16 was stored at (4±1)℃ for 14 days. To avoid interference from DES in the measurement results, the sample solution was dialyzed (MWCO 3000Da) and then subjected to circular dichroism spectroscopy to assess drug stability.

[0188] like Figure 14 As shown, due to the presence of the α-helical structure in UOX, the DES(2:1)-UOX complex exhibits double negative valley peaks at approximately 207 nm and 222 nm. The ellipticity of DES(2:1)-UOX is not significantly different from that of free UOX, indicating that DES(2:1)-UOX has good stability and UOX retains its original activity.

[0189] Example 18: In vitro release study of DES(2:1)-UOX

[0190] To investigate the release of DES(2:1)-UOX in vivo, the cumulative release of UOX under simulated intestinal fluid conditions (pH 6.8) was determined using an in vitro simulation method. The specific steps were as follows: DES(2:1) aqueous solution (10% by volume of DES2:1) was added to the 8 mg / mL FITC-UOX solution prepared in Example 16, and the mixture was vortexed to prepare a DES-FITC-UOX solution (UOX concentration of 2 mg / mL). Six mL of the DES(2:1)-FITC-UOX solution was divided into three equal portions and placed in dialysis bags (MWCO: 3000 Da). These bags were then placed in phosphate buffer (pH 6.8, i.e., simulated intestinal fluid) in triplicate and subjected to an in vitro release experiment in a constant temperature water bath shaker (37°C, 100 rpm). Samples of 500 μL were taken at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 hours after the start of the experiment, and an equal volume of fresh release medium was added simultaneously. The fluorescence intensity of UOX at each time point was measured using a Cytation5 multimode microplate detection and cell imaging system at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and the cumulative release was calculated.

[0191] like Figure 15 As shown, DES(2:1)-UOX was slowly released in simulated intestinal fluid, reaching 80% at 6 hours and reaching release equilibrium at 12 hours.

[0192] Example 19: Oral bioavailability study of DES(2:1)-UOX

[0193] Preparation of DES(2:1)-UOX solution: Dissolve 5 mg of urate oxidase (UOX) in 1 mL of distilled water and mix well. Add the above UOX solution dropwise to 1 mL of DES2:1 and vortex to mix well to obtain DES(2:1)-UOX with a drug concentration of 2.5 mg / mL.

[0194] Six male SD rats (weighing approximately 250±20g) were randomly divided into two groups according to body weight: a subcutaneous injection group (Free-UOX) and an intestinal injection group (DES(2:1)-UOX). The subcutaneous injection group received UOX aqueous solution (2U / kg), while the intestinal injection group received DES(2:1)-UOX (200U / kg). Rats were fasted for 24 hours prior to the experiment. Blood samples (0.5mL / sample) were collected from the medial canthal venous plexus of the rats at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 hours of the experiment. The uric acid-lowering activity of UOX in the plasma samples was determined by a uricase activity assay, and the plasma concentration of the drug was calculated to evaluate the oral bioavailability of DES(2:1)-UOX.

[0195] Table 14: Pharmacokinetic parameters of the oral urate oxidase formulation of the present invention in rats.

[0196]

[0197] UOX plasma concentration-time curves and pharmacokinetic parameters in rats are as follows: Figure 16 As shown in Table 14, the relative bioavailability of the DES(2:1)-UOX formulation was 1.66%. These results indicate that DES2:1 significantly improves the oral bioavailability of UOX.

Claims

1. A deep eutectic solvent, characterized in that: The deep eutectic solvent is an organic molten salt composed of anionic and cationic components; wherein the anionic component is selected from organic acids, polyols or sugars that can act as hydrogen bond donors; the cationic component is selected from quaternary ammonium salts and zwitterionic surfactants that can act as hydrogen bond acceptors; and the molar ratio of the cationic component to the anionic component is 1:4 to 1:0.

5.

2. The deep eutectic solvent according to claim 1, characterized in that: The anionic component is geraniol; the cationic component is choline or its bicarbonate.

3. The deep eutectic solvent according to claim 1 or 2, characterized in that: The molar ratio of the cationic component to the anionic component is 1:

2.

4. A macromolecular pharmaceutical composition prepared based on deep eutectic solvent technology, characterized in that: It includes an external enteric protective layer and drug-loaded composite particles or drug-loaded composite microspheres encapsulated inside the enteric protective layer; the drug-loaded composite particles or drug-loaded composite microspheres are prepared from a deep eutectic solvent-macromolecule drug delivery system and other excipients; the deep eutectic solvent-macromolecule drug delivery system is made from a biological macromolecule drug and the deep eutectic solvent as described in any one of claims 1-3.

5. The macromolecular pharmaceutical composition according to claim 4, characterized in that: The macromolecular drug composition is an oral dosage form, which may be an enteric-coated capsule, enteric-coated tablet, enteric-coated granule, or enteric-coated microsphere.

6. The macromolecular pharmaceutical composition according to claim 4, characterized in that: The excipients are one or a combination of fillers, lubricants, and disintegrants; the fillers are lactose, mannitol, or sorbitol, preferably mannitol; the lubricant is magnesium stearate; and the disintegrant is starch.

7. The macromolecular pharmaceutical composition according to claim 4, characterized in that: The enteric protective layer is an enteric capsule shell and an enteric coating layer.

8. The macromolecular pharmaceutical composition according to claim 4, characterized in that: The aforementioned biological macromolecular drugs are proteins, sugars or polysaccharides, polypeptides, and enzymes; preferably, low molecular weight heparin, calcitonin, or uricase.

9. A method for preparing the macromolecular pharmaceutical composition according to claim 4, characterized in that: include: Step (1): Place the cationic component solution in a water bath at 35℃~45℃, and add the anionic component dropwise to the cationic component solution under stirring until no carbon dioxide is released. Step (2): After the reaction is complete, the solvent in the reaction solution is removed by vacuum evaporation. Step (3): Place the product in a vacuum drying oven to dry, and obtain a deep eutectic solvent; Step (4): Dissolve the biomacromolecule drug in distilled water to obtain an aqueous solution of the biomacromolecule drug. Add the aqueous solution of the biomacromolecule drug dropwise to the eutectic solvent and mix evenly to obtain the eutectic solvent-macromolecule drug delivery system. Step (5): Prepare drug-loaded composite particles or drug-loaded composite microspheres by combining the deep eutectic solvent-macromolecule drug delivery system with excipients; encapsulate the drug-loaded composite particles or drug-loaded composite microspheres with an enteric protective layer to prepare enteric capsules, enteric tablets, enteric granules or enteric microspheres.

10. The method for preparing the macromolecular pharmaceutical composition according to claim 9, characterized in that: In step (1), the temperature of the water bath is 40°C; In step (2), the temperature of the reduced pressure evaporation is 50℃~60℃; In step (3), the drying temperature is 50℃~60℃ and the drying time is 24~48 hours; In step (4), the concentration of the aqueous solution of the biomacromolecule drug is 1 to 100 mg / mL; the volume ratio of the aqueous solution of the biomacromolecule drug to the deep eutectic solvent is 1:20 to 1:0.5, preferably 1:10 to 3:2, and more preferably 1:1.5 to 1:

1. In step (5), when the dosage form of the macromolecular drug composition is enteric-coated capsules, the deep eutectic solvent-macromolecular drug and filler are mixed and wet granulated to prepare drug-loaded composite particles; the drug-loaded composite particles are encapsulated into enteric-coated capsules; wherein, the ratio of the deep eutectic solvent to the filler is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg; When the dosage form of the macromolecular drug composition is an enteric-coated tablet, the eutectic solvent-macromolecular drug is mixed with filler and disintegrant, and wet granulation is performed to prepare drug-loaded composite particles; then, it is mixed with a lubricant and compressed into tablets using a tablet press to obtain tablet cores; the enteric coating components are sequentially dissolved in ethanol to prepare a coating solution; the tablet cores are coated using a coating machine and dried to form an enteric coating layer, thus preparing macromolecular drug enteric-coated tablets; wherein, the ratio of the total amount of eutectic solvent to filler and disintegrant is 5:10 to 7:

1. The filler and disintegrant have a mass ratio of 0 μL / mg, preferably 6:10 μL / mg; the mass ratio of the filler to the disintegrant is 8:1 to 10:1, preferably 9:1; the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol at a mass ratio of 6.5 to 7.5:6.5 to 7.5:3.5 to 4.5:0.8 to 1.2:1, and the concentration of polyacrylic acid resin II in the coating solution is 1 to 10 mg / mL, preferably 1 mg / mL; When the dosage form of the macromolecular drug composition is enteric-coated granules, the eutectic solvent, macromolecular drug, and filler are mixed and wet-granulated to prepare drug-loaded composite granules; the components of the enteric coating are sequentially dissolved in ethanol to prepare a coating solution; the drug-loaded composite granules are coated using a coating machine and dried to form an enteric coating layer to prepare macromolecular drug enteric-coated granules; wherein, the ratio of the eutectic solvent to the filler is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg; the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80, and dimethyl phthalate in anhydrous ethanol at a mass ratio of 6.5 to 7.5:6.5 to 7.5:3.5 to 4.5:0.8 to 1.2:1, and the concentration of polyacrylic acid resin II in the coating solution is 1 to 10 mg / mL, preferably 1 mg / mL; When the dosage form of the macromolecular drug composition is enteric-coated microcapsules, the eutectic solvent, macromolecular drug, and filler are mixed, extruded into thin strips using an extruder, granulated and sphericalized using a spheronizer, and then dried to obtain drug-loaded composite microcapsules; the components of the enteric coating are sequentially dissolved in ethanol to prepare a coating solution; the drug-loaded composite microcapsules are coated using a coating machine, dried to form an enteric coating layer, and the oral enteric-coated microcapsules of the macromolecular drug are prepared; wherein, the eutectic solvent and filler are used... The mass ratio is 5:10 to 7:10 μL / mg, preferably 6:10 μL / mg; the coating solution is prepared by dissolving polyacrylic acid resin II, polyacrylate III, castor oil, polysorbate 80 and dimethyl phthalate in anhydrous ethanol at a mass ratio of 6.5 to 7.5:6.5 to 7.5:3.5 to 4.5:0.8 to 1.2:1, and the concentration of polyacrylic acid resin II in the coating solution is 1 to 10 mg / mL, preferably 1 mg / mL.

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