Application of biotin-based drugs and application of biotin sustained-release formulations

Abstract

This application relates to the field of biomedical technology, and in particular to the application of biotin-based drugs and biotin sustained-release formulations. Biotin-based drugs are used to prepare medications that promote cartilage formation, slow the progression of osteoarthritis, and inhibit cartilage loss, and can effectively relieve and treat osteoarthritis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of biotin-based drugs and biotin sustained-release formulations. Background Technology

[0002] Osteoarthritis (OA) is a joint disease characterized by the gradual wear and tear and destruction of articular cartilage. It is often accompanied by bone hyperplasia, synovitis, and other manifestations, leading to joint pain and functional impairment. Current treatments mainly aim to relieve symptoms and slow disease progression, including non-pharmacological treatments (such as exercise therapy and physical therapy), pharmacological treatments (such as nonsteroidal anti-inflammatory drugs and intra-articular injections), and surgical treatments (such as joint replacement).

[0003] Intra-articular injection is one of the most common treatments for osteoarthritis (OA) in clinical practice. However, current conventional injections of drugs such as sodium hyaluronate have short-lived effects, often failing to meet patient expectations, and the associated pain from frequent injections results in very low patient compliance. Furthermore, conventional drugs such as dexamethasone and flurbiprofen, carried in cross-linked hyaluronic acid (HA), only provide anti-inflammatory and analgesic effects, with limited efficacy in cartilage repair. Summary of the Invention

[0004] Based on this, the first aspect of this application provides the use of a biotinylated drug in the preparation of a drug that promotes cartilage formation, slows the progression of osteoarthritis, or inhibits cartilage loss.

[0005] The second aspect of this application provides the use of a biotinylated drug in the preparation of a medicament for the treatment and / or prevention of osteoarthritis.

[0006] The third aspect of this application provides the use of biotin-release formulations in the preparation of medicaments that promote cartilage formation, slow the progression of osteoarthritis, or inhibit cartilage loss.

[0007] The fourth aspect of this application provides the use of biotin-release formulations in the preparation of medicaments for the treatment and / or prevention of osteoarthritis.

[0008] In some embodiments, the biotin-release formulation includes sustained-release particles, an intermediate layer, and an outer layer; the sustained-release particles include a sustained-release material and a biotinylated drug; the intermediate layer coats the sustained-release particles and includes cationic liposomes; the outer layer coats the intermediate layer and includes cross-linked hyaluronic acid.

[0009] In some embodiments, the preparation method of the biotin sustained-release formulation includes the following steps:

[0010] Prepare sustained-release granules, wherein the sustained-release granules comprise a sustained-release material and a biotinylated drug;

[0011] An intermediate layer is coated onto the sustained-release particles, the intermediate layer comprising cationic liposomes;

[0012] An outer layer is coated on the intermediate layer, and the outer layer includes cross-linked hyaluronic acid.

[0013] Compared with traditional solutions, this application has the following advantages:

[0014] This application, through drug screening, discovered that biotin has the function of promoting cartilage differentiation, and the repair of cartilage wear in articular cartilage is a key challenge in the incurable nature of arthritis. Therefore, this application uses biotin-based drugs to prepare medications that promote cartilage formation, slow the progression of osteoarthritis, and inhibit cartilage loss. Furthermore, this application uses biotin-based drugs to prepare medications for the treatment and / or prevention of osteoarthritis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Results of Western blot analysis of cellular proteins and results of toluidine blue staining test;

[0017] Figure 2 The results are from a cellular immunofluorescence assay.

[0018] Figure 3 Results of oral drug treatment of the knee joint in mice;

[0019] Figure 4 Results of human knee cartilage explant culture;

[0020] Figure 5 Results of drug injection into the knee joint of rats. Detailed Implementation

[0021] The present application will be further described in detail below with reference to specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0023] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0024] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0025] In this application, the terms "optionally," "optionally," and "optional" refer to options that are optional, meaning they can be selected from either "with" or "without." If multiple "optional" options appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" option is independent.

[0026] In this application, the terms "first aspect," "second aspect," "third aspect," and "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," and "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0027] In this application, numerical intervals (i.e. numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the above-mentioned numerical intervals are considered continuous, and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as every value between the two numerical endpoints.

[0028] Biotin, also known as vitamin B7, is a water-soluble vitamin. Biotin is essential for a variety of physiological functions. It is considered to have been discovered by the Hungarian-American biochemist Paul Gyorgy, who first isolated it from egg yolks in 1936. As a coenzyme for carboxylases, biotin participates in the metabolism of carbohydrates, fats, and proteins, promoting energy production and nutrient synthesis. Biotin also plays a significant role in the health of skin, hair, and nails, maintaining their structure and function. Furthermore, it supports nervous system health, regulates blood sugar levels, and plays an important role in pregnancy and infant development. Currently, biotin is used for various indications, both as a dietary supplement and to treat certain medical conditions such as diabetes, eczema, peroneal muscular dystrophy, and adrenoleukodystrophy. Previous studies have reported that biotin deficiency exacerbates inflammation, but research on its specific mechanisms in arthritis is lacking, and there are no reports of biotin treatment for osteoarthritis (OA).

[0029] This application, through drug screening, discovered that biotin has the function of promoting cartilage differentiation, and the repair of cartilage wear in articular cartilage is a key challenge in the treatment of arthritis. Therefore, this application utilizes biotin-based drugs in the preparation of drugs that promote cartilage formation, slow the progression of osteoarthritis, and inhibit cartilage loss. The first aspect provides the use of biotin-based drugs in the preparation of drugs that promote cartilage formation, slow the progression of osteoarthritis, or inhibit cartilage loss. The second aspect provides the use of biotin-based drugs in the preparation of drugs for the treatment and / or prevention of osteoarthritis.

[0030] Biotin drugs, including one or more of biotin, biotin derivatives, and biotin analogs, can effectively relieve and treat osteoarthritis.

[0031] A third aspect of this application provides the use of a biotin sustained-release formulation in the preparation of a medicament that promotes cartilage formation, slows the progression of osteoarthritis, or inhibits cartilage loss. A fourth aspect of this application provides the use of a biotin sustained-release formulation in the preparation of a medicament for the treatment and / or prevention of osteoarthritis.

[0032] In one embodiment, the biotin sustained-release formulation includes a core, an intermediate layer, and an outer layer; the core includes sustained-release particles and a biotin-like drug loaded on the sustained-release particles; the intermediate layer encapsulates the core and includes cationic liposomes; and the outer layer encapsulates the intermediate layer and includes cross-linked hyaluronic acid.

[0033] This embodiment provides a sustained-release formulation using biotin as the active pharmaceutical ingredient. The formulation includes sustained-release particles, each comprising a sustained-release material and a biotin-based drug. An intermediate layer encapsulating the sustained-release particles includes cationic liposomes, and an outer layer encapsulating the intermediate layer includes cross-linked hyaluronic acid (HA). This provided biotin sustained-release formulation can carry a high content of biotin in a small volume, effectively relieving and treating osteoarthritis. Furthermore, through the primary sustained-release effect of the sustained-release particles and the auxiliary sustained-release effects of the cationic liposome layer and cross-linked HA, it achieves sustained and controlled drug release.

[0034] In some examples, the sustained-release material includes a polymeric sustained-release material. Optionally, the weight-average molecular weight of the polymeric sustained-release material is 10,000 Da to 100,000 Da. For example, the weight-average molecular weight of the polymeric sustained-release material is 10,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 90,000 Da, or 100,000 Da. Preferably, it is 10,000 Da to 80,000 Da. Wherein, Da represents Dalton. Optionally, the polymeric sustained-release material is selected from one or more of sodium alginate, chitosan, polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate copolymer, polyorthoester, polyanhydride, and cross-linked hyaluronic acid (HA). Optionally, the polymeric sustained-release material is selected from PLGA. PLGA is a biodegradable functional polymeric organic compound formed by the random polymerization of two monomers—lactic acid and glycolic acid. It possesses good biocompatibility, is non-toxic, and exhibits excellent encapsulation and film-forming properties. PLGA, when used in conjunction with biotin, demonstrates excellent stability and drug release. The weight-average molecular weight of PLGA is 50,000 Da to 80,000 Da, ensuring good degradation controllability, balancing degradation rate and drug loading, and achieving long-lasting sustained release for 21 to 28 days. Optionally, the mass ratio of biotinylated drug to polymeric sustained-release material is (0.1 to 2):(10 to 50). Further optionally, the mass ratio of biotinylated drug to polymeric sustained-release material is 1:(50 to 100). For example, mass ratios of 1:50, 1:60, 1:70, 1:80, 1:90, and 1:100 are used. The above-mentioned range is beneficial for balancing drug load and release kinetics, resulting in sustained-release formulations with good stability, long in vivo residence time, good drug release properties, and high bioavailability. Experimental verification shows that when biotin is used as the biotinylate and PLGA is used as the sustained-release material, a 1:50 ratio of the two in sustained-release particles can effectively reduce the burst release rate (<5%) and extend the release period to 28 days.

[0035] In other examples, the sustained-release particles comprise small-molecule sustained-release materials selected from lipid compounds, preferably lipid-like substances, more preferably phospholipids and / or steroids. The phospholipids may be one or more of lecithin, sphingomyelin, cephalin, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. The steroids may be one or more of cholesterol, sitosterol, stigmasterol, ergosterol, and their derivatives.

[0036] The molecular weight of the sustained-release material can be selected based on whether it is for palliative or therapeutic use. In palliative use, relatively low doses are administered at relatively low intervals over a long period, requiring a longer sustained-release period for biotinylated drugs. In this case, a high molecular weight sustained-release material can be chosen. In therapeutic use, sometimes relatively high doses are administered at relatively short intervals until disease progression is slowed or stopped, requiring a relatively short sustained-release period for biotinylated drugs. In this case, a low molecular weight sustained-release material can be chosen.

[0037] Optionally, the sustained-release particles further include an emulsifier. The emulsifier is a hydrophilic emulsifier. An emulsifier is a substance that can improve the surface tension between various constituent phases in an emulsion, thereby forming a uniform and stable dispersion system or emulsion. In this embodiment, an emulsifier is used to prepare a water-in-oil-in-water double emulsion of biotinylate and sustained-release material. The oil film barrier of the emulsion droplets delays drug release, avoiding the use of organic solvents, thus achieving a long-acting and safe formulation that can be directly injected. The emulsifier is selected from one or more of polyvinyl alcohol, Tween 80, PEG-100 stearate, glyceryl stearate, arachidonic acid alcohol, fatty alcohol polyoxyethylene ether, glyceryl behenate, cetearyl glucoside, and poloxamer 407. Preferably, the emulsifier is poloxamer 407, which has thermosensitive properties and forms micelles at 37°C, further enhancing emulsion stability and sustained-release performance, while reducing the use of organic solvents and improving safety. Optionally, the emulsifier is added at a mass ratio of 3% to 7% of the sustained-release material. For example, the emulsifier is added at a mass ratio of 3%, 5%, or 7% of the sustained-release material.

[0038] Optionally, the particle size of the sustained-release particles is 200 nm ± 50 nm.

[0039] The cationic liposomes encapsulate the core, providing active charge targeting capability. Optionally, the cationic liposomes comprise (2,3-dioleoylpropyl)-trimethylamine, which carries a positive charge and has a high affinity for negatively charged cell membranes (such as chondrocytes). Furthermore, (2,3-dioleoylpropyl)-trimethylamine can bind to biotin / PLGA sustained-release particles via electrostatic interactions, providing cell targeting and a secondary sustained-release barrier, with an encapsulation efficiency >90%. Optionally, the mass ratio of the intermediate layer to the sustained-release particles is 1:(2.5~4.5).

[0040] Optionally, the intermediate layer further includes a zinc ion-polyphenol complex. The zinc ion-polyphenol network comprises Zn 2+ -EGCG (epigallocatechin gallate) complex. The Zn... 2+ -EGCG complexes, acting as auxiliary functional materials, enhance pH-responsive release through metal coordination, accelerating drug release in the osteoarthritis microenvironment (pH 6.5–7.0). Zn 2+ In EGCG complexes, Zn 2+ The mass ratio of zinc ion-polyphenol complex to cationic liposomes is (0.3~0.7):10. For example, the mass ratio is 0.3:10, 0.5:10, or 0.7:10. Optionally, the mass ratio of zinc ion-polyphenol complex to cationic liposomes is 1:(8~12). For example, 1:8, 1:10, or 1:12.

[0041] Optionally, the outer layer includes cross-linked hyaluronic acid (HA). HA, a natural component of synovial fluid, is cross-linked to form HA with excellent viscoelasticity and biocompatibility. Cross-linked HA, as a biomaterial with a stable network structure obtained through chemical cross-linking modification, exhibits multi-dimensional advantages in the treatment of osteoarthritis. With its excellent biocompatibility and biodegradability, it can not only serve as a drug carrier for long-term sustained release and targeted delivery—encapsulating drug molecules through a cross-linked network, significantly prolonging the intra-articular drug action time and precisely targeting diseased cartilage and synovial tissue—but also form a lubricating protective layer within the joint, directly inhibiting inflammatory cell infiltration and factor release, thereby effectively alleviating synovitis symptoms. Furthermore, cross-linked HA, by mimicking the lubricating and buffering properties of natural cartilage, provides a microenvironment conducive to chondrocyte proliferation and differentiation, promoting collagen synthesis and cartilage tissue repair. Moreover, its increased mechanical strength provides more durable mechanical support to the joint, significantly improving joint function and reducing wear. Existing research has confirmed that cross-linked HAs, such as HA-Cross and HA-Link, can reduce inflammatory factor levels and promote cartilage repair in a rat model of knee osteoarthritis. Clinical trials have also shown that they can relieve pain and improve joint function with good safety. Therefore, by integrating multiple mechanisms such as controlled drug release, anti-inflammatory repair, and biomechanical protection, cross-linked HAs provide a comprehensive solution with promising clinical applications for the precision treatment of osteoarthritis.

[0042] Optionally, the degree of cross-linking of the cross-linked HA is 15%~25%, which is beneficial for balancing mechanical strength and degradation rate. Optionally, the weight-average molecular weight of the cross-linked HA is 1.0MDa~1.5MDa, ensuring good mechanical strength (compressive modulus 25kPa~35kPa) and controllable degradation (in vivo retention time 3~4 weeks). Optionally, the cross-linking agent of the cross-linked HA includes divinyl sulfone (DVS). As a smart and stable outer barrier, the cross-linked HA can intelligently regulate drug release in response to the osteoarthritis microenvironment (pH 6.5~7.0), significantly improving the sustained-release performance and safety of the dosage form. Optionally, the mass ratio of the outer layer to the sustained-release particles is 1:(1.3~1.8).

[0043] The above-mentioned material system innovation and process optimization of biotinylate drugs have been carried out. Due to the limitations of traditional single materials, a multi-level composite sustained-release system has been constructed. Through a three-level sustained-release mechanism and intelligent material design, the half-life has been extended from 2 hours to 28 days, which significantly improves the therapeutic effect and clinical application value of the drug.

[0044] In one embodiment, the method for preparing a biotin sustained-release formulation includes the following steps:

[0045] S10. Prepare sustained-release granules, wherein the sustained-release granules include sustained-release materials and biotinylated drugs.

[0046] Optionally, the sustained-release granules also include an emulsifier. The preparation method of the sustained-release granules includes the following steps: dissolving the sustained-release material and biotinylated drug in an organic solvent, adding the emulsifier, and forming a uniform emulsion by ultrasonic treatment; removing the organic solvent by vacuum rotary evaporation of the emulsion to form solid microspheres, which are the sustained-release granules.

[0047] Optionally, the boiling point of the organic solvent is not higher than 50°C. This ensures that the solvent can be evaporated at room temperature or with slight heating during subsequent removal processes, avoiding purification operations such as distillation and extraction. This simplifies the preparation process while maintaining effectiveness. Optionally, the organic solvent is selected from one or more of acetone, chloroform, methylamine, dimethylamine, diethyl ether, pentane, dichloromethane, and carbon disulfide. In this embodiment, the organic solvent is selected from dichloromethane. Dichloromethane has a low boiling point (39.6°C), good volatility, low toxicity, and is easily removed by evaporation, minimizing toxic side effects.

[0048] Optionally, 10 mg to 50 mg of the sustained-release material and 0.1 mg to 2 mg of the biotinylate are dissolved in 0.1 mL to 2 mL of organic solvent. The sustained-release material can be 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, or 50 mg. The biotinylate can be 0.1 mg, 0.5 mg, 1 mg, 1.5 mg, or 2 mg. The organic solvent can be 0.1 mL, 0.5 mL, 1 mL, 1.5 mL, or 2 mL. Understandably, the sustained-release material and biotinylate are dissolved in the organic solvent by stirring or sonication.

[0049] In this embodiment, the biotinylated drug is biotin, and the sustained-release material is PLGA. Each 10mg~50mg PLGA and 0.1mg~2mg biotin are dissolved in 0.1mL~2mL of dichloromethane.

[0050] Optionally, the emulsifier is added at a mass ratio of 3% to 7% of the sustained-release material.

[0051] In this embodiment, the emulsifier is poloxamer 407. Poloxamer 407 reduces the interfacial tension between oil and water, enabling PLGA and biotin to form a stable O / W (oil-in-water) emulsion in dichloromethane, thereby achieving a high encapsulation rate (>90%). The mass of poloxamer 407 added accounts for 3% to 7% of the mass of PLGA.

[0052] Optionally, the parameters of the ultrasonic treatment are controlled to ensure that the microsphere size in the emulsion is 200 nm ± 50 nm. This particle size is beneficial for cellular uptake and in vivo distribution. Optionally, the ultrasonic treatment power is 300 W to 400 W, and the time is 20 min to 30 min. An ice bath is maintained during the ultrasonic treatment to avoid overheating.

[0053] The emulsion was placed in a rotary evaporator and subjected to reduced pressure rotary evaporation at 40°C and 0.09 MPa to remove the organic solvent. These conditions prevented degradation of biotin by high temperatures and ensured that the residual organic solvent content was <0.005% (compliant with ICH Q3C standards). After evaporation, the solution solidified into solid microspheres, which were collected by centrifugation and washed with deionized water to remove residual organic solvent.

[0054] S20. Coating the sustained-release particles with an intermediate layer, the intermediate layer comprising cationic liposomes.

[0055] Optionally, coating the sustained-release particles with an intermediate layer includes the following steps: dissolving cationic liposomes in a solvent to obtain a cationic liposome solution; rotary evaporating the cationic liposome solution to obtain a lipid film; mixing the lipid film and phosphate buffer to obtain a liposome suspension; filtering and centrifuging the liposome suspension to obtain a liposome stabilizing solution; mixing the sustained-release particles and the liposome stabilizing solution, and centrifuging.

[0056] Optionally, the intermediate layer further includes a zinc ion-polyphenol complex, which coats the sustained-release particles, comprising the following steps: dissolving cationic liposomes and polyphenol compounds in a solvent to obtain a cationic liposome composite solution; rotary evaporating the cationic liposome composite solution to obtain a lipid composite film; mixing the lipid film, zinc ion solution, and phosphate buffer to obtain a liposome composite suspension; filtering and centrifuging the liposome composite suspension to obtain a liposome composite stabilized solution; and mixing the sustained-release particles and the liposome composite stabilized solution, followed by centrifugation.

[0057] Optionally, the solvent is selected from chloroform. In the cationic liposome solution, the concentration of cationic liposomes is 5 mg / mL to 15 mg / mL. In the cationic liposome composite solution, the total concentration of cationic liposomes and polyphenolic compounds is 5 mg / mL to 15 mg / mL. The cationic liposome solution or cationic liposome composite solution is placed in a rotary evaporator, the water bath temperature of the rotary evaporator is set to 36-42°C, and the vacuum pump is turned on to evacuate for 30-40 minutes. The resulting milky white coating is the lipid film or lipid composite film.

[0058] Optionally, the zinc ion-polyphenol complex is selected from Zn 2+- EGCG complex, the polyphenol compound is selected from EGCG. Zinc ion solution is selected from ZnCl2 aqueous solution, concentration 1mg / mL~2mg / mL. Phosphate buffer mass fraction is 40%~60%. For example, mass fraction of 40%, 50%, 60%. After adding phosphate buffer, sonicate for 40 to 50 minutes to obtain a cloudy white liquid, which is the liposome suspension or liposome complex suspension. The liposome suspension consists of cationic liposomes suspended in phosphate buffer. The liposome complex suspension consists of cationic liposomes and zinc ion-polyphenol complex suspended in phosphate buffer.

[0059] The liposome suspension or liposome complex suspension was filtered through a filter membrane with a pore size of 100 nanometers. The filtrate was centrifuged at 18,000 rpm to 22,000 rpm for 30 to 40 minutes, and repeated 5 to 10 times to obtain a clear and stable liquid with uniform particle size, which is the liposome stable liquid or liposome complex stable liquid.

[0060] Liposome stabilizing solution or liposome composite stabilizing solution is mixed with sustained-release particles at a volume ratio of 1:(1.5~3). After sonication for 3 to 5 minutes, the mixture is centrifuged at 20,000 rpm to 25,000 rpm for 15 to 20 minutes. The supernatant is removed, and the precipitate is resuspended with an equal volume of phosphate solution. This process is repeated 1 to 2 times to obtain sustained-release particles with an intermediate layer.

[0061] The pH-responsive release rate of the slow-release particles encapsulated in the intermediate layer was tested. The results showed that the release rate was 20% to 30% higher than that of the slow-release particles under pH 6.5-7.0 conditions after the intermediate layer coating. The encapsulation efficiency was maintained at >90%, and the particle size was still controlled within 200±50 nm by dynamic light scattering method.

[0062] S30. An outer layer is coated on the intermediate layer, the outer layer comprising cross-linked HA.

[0063] Optionally, covering the intermediate layer with an outer layer includes the following steps:

[0064] Sodium hyaluronate was dissolved in phosphate-buffered saline (PBS), a cross-linking agent was added, and a cross-linking reaction occurred. The reaction solution was purified by dialysis to obtain a cross-linked HA solution.

[0065] A mixed cross-linked HA solution and a slow-release particle encapsulated in an intermediate layer are adsorbed and then solidified.

[0066] Optionally, the pH of the phosphate buffer solution is 7.4–10.0. Sodium hyaluronate is dissolved in the phosphate buffer solution to prepare a solution with a sodium hyaluronate content of 1%–3% (w / v, mg / mL). The crosslinking agent is selected from DVS. After adding the crosslinking agent, the pH of the solution is adjusted to 9.5–10.0. Within this range, the solution is in an alkaline environment, which promotes ether bond formation. The crosslinking reaction is stirred during the process, and the progress is controlled by viscosity monitoring. The amount of crosslinking agent, the temperature of the crosslinking reaction, and the time are controlled to ensure that the degree of crosslinking is within the ideal range of 15%–25%, which is beneficial for balancing mechanical strength and degradation rate. At this time, the viscosity of the crosslinked HA at 25°C is 500 mPa·s–800 mPa·s, and the weight-average molecular weight is 1.0 MDa–1.5 MDa, indicating that the crosslinked network is fully formed. Optionally, the mass of the crosslinking agent is 0.5%–1% of the mass of sodium hyaluronate. The temperature of the crosslinking reaction is room temperature, which is between 10°C and 40°C, for example, 25°C. The cross-linking reaction takes 6 to 8 hours.

[0067] The reaction solution obtained from the cross-linking reaction was placed in a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed with deionized water for 48 hours to thoroughly remove unreacted DVS and byproducts, ensuring biosafety. It was then sterilized and stored at 4°C for later use. Gamma ray sterilization (dose 15 kGy~25 kGy) was used to avoid high temperatures damaging the HA structure. After sterilization, the cross-linked HA was stored at 4°C to prevent freezing damage to its three-dimensional network. This step optimized the cross-linking efficiency and purity, controlling the cross-linking agent residue to <0.005% and achieving a cell viability >98%.

[0068] The cross-linked HA solution and the sustained-release particles encapsulated in the intermediate layer were mixed at a volume ratio of 1:(0.8~1.2). After mixing, the mixture was adsorbed at 2~6℃ for 1 hour to 3 hours, and then cured at 35~40℃ for 10 minutes to 30 minutes to complete integration. Adsorption ensures that the HA network initially encapsulates the intermediate layer, and the curing stage forms a stable gel barrier at physiological temperatures, enhancing mechanical strength (compressive modulus 25kPa~35kPa) and sustained-release performance (in vivo retention time 3~4 weeks).

[0069] The preparation method of the biotin sustained-release formulation provided by the above embodiments is simple, convenient and quick. The prepared biotin sustained-release formulation has good stability, long in vivo retention time, good drug release and high bioavailability, which is conducive to the promotion and application of industrial production.

[0070] In this embodiment, the biotin sustained-release formulation is an injectable. The sustained-release formulation of this application can be administered by injection, including intravenous injection, local injection, and intra-articular injection. This embodiment achieves a long-acting sustained-release injectable through a multi-layered core-shell structure, which can be used to treat osteoarthritis and other chronic diseases requiring long-term medication. The sustained-release formulation provided in this embodiment can be injected directly without the aid of organic solvents, reducing adverse reactions and safety incidents caused by organic solvents, solving the dilemma of non-biotin injectable formulations, achieving local drug delivery, and having a long retention time in the body, eliminating the need for repeated injections, reducing the cost of multiple administrations, and lowering patient pain and irritation.

[0071] Administration of the biotin extended-release formulation or extended-release formulation of this application may include additional second agents, such as organic diphosphates, chemotherapeutic agents, radiopharmaceuticals, TNF-antagonists, nonsteroidal anti-inflammatory agents, steroids, antioxidants, angiogenesis inhibitors, matrix metalloproteinase inhibitors, vitamins, selective estrogen receptor modulators, estrogen-progesterone, androgens, calcitonin, antibiotics, cathepsin K inhibitors, inhibin, integrin receptor antagonists, osteoblast anabolic metabolites or selective serotonin reuptake inhibitors, glucosamine or mixtures thereof.

[0072] It should be noted that the diseased “subject,” “patient,” or “animal” treated by the method of this application can be a human or a non-human mammal, and therefore can be a human and a dog, cat, mouse, rat, cow, sheep, pig, goat, or primate, and may include laboratory animals, livestock, and domestic animals.

[0073] The following description is further illustrated with specific embodiments and comparative examples. Unless otherwise specified, the raw materials involved in the following specific embodiments and comparative examples are all commercially available. Unless otherwise specified, the instruments used are all commercially available. Unless otherwise specified, the processes involved are conventionally selected by those skilled in the art.

[0074] Example 1

[0075] This embodiment provides a biotin sustained-release formulation, its preparation method, and the steps of the preparation method are as follows:

[0076] Step 1: Preparation of sustained-release granules

[0077] 50 mg PLGA and 1 mg biotin were added to 2 mL of dichloromethane and magnetically stirred until dissolved. Then, 2.5 mg poloxamer 407 was added, and the mixture was ultrasonically treated to form a homogeneous emulsion. The ultrasonic treatment power was 350 W for 25 min, and the microspheres in the emulsion had a particle size of 200 nm ± 50 nm. The emulsion was then placed in a rotary evaporator and subjected to reduced pressure rotary evaporation at 40 °C and 0.09 MPa to remove dichloromethane. After evaporation, the solution solidified into solid microspheres, which were collected by centrifugation (10,000 rpm × 10 min) and washed with deionized water to remove residual organic solvent, yielding sustained-release particles.

[0078] Step 2, Wrap the intermediate layer

[0079] 160 mg of (2,3-dioleoyl-propyl)-trimethylamine and 46 mg of EGCG were dissolved in 20 mL of chloroform to obtain a cationic liposome complex solution. The cationic liposome complex solution was placed in a rotary evaporator, and the water bath temperature was set to 40 °C. After vacuuming for 30 minutes, a milky white lipid complex film formed in the reactor. 9.5 mL of 50% phosphate buffer and 0.5 mL of 1 mg / mL ZnCl2 aqueous solution were added to the lipid complex film, and the mixture was sonicated for 40 minutes to obtain a cloudy white liposome complex suspension, consisting of cationic liposomes and zinc ion-polyphenol complexes suspended in phosphate buffer. The liposome complex suspension was filtered through a 100 nm filter membrane, and the filtrate was centrifuged at 18,000 rpm for 30 minutes, repeated 8 times, to obtain a clear, stable liposome complex solution. The liposome composite stabilizing solution was mixed with the sustained-release particles from step 1 at a volume ratio of 1:2. After sonication for 5 minutes, the mixture was centrifuged at 20,000 rpm for 15 minutes to remove the supernatant. The precipitate was resuspended with an equal volume of phosphate solution. This process was repeated twice to obtain sustained-release particles with an intermediate layer.

[0080] Step 3: Coat the outer layer

[0081] Sodium hyaluronate was dissolved in PBS (pH 9.5) to prepare a 2% (w / v) solution. 0.5% (by weight of sodium hyaluronate) of the cross-linking agent DVS was added, and the pH was adjusted to 9.5. A cross-linking reaction occurred at room temperature. After 6 hours, the degree of cross-linking was 15%–25%, yielding the reaction solution. The reaction solution was placed in a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed with deionized water for 48 hours to thoroughly remove unreacted DVS and byproducts, ensuring biosafety. The solution was sterilized and stored at 4°C for later use. Gamma ray sterilization (dose 15 kGy–25 kGy) was used to avoid high-temperature damage to the HA structure. After sterilization, the cross-linked HA was stored at 4°C to prevent freezing damage to its three-dimensional network, resulting in a cross-linked HA solution. The cross-linked HA solution was mixed with the encapsulated sustained-release particles from step 2 at a 1:1 volume ratio. After adsorption at 4°C for 2 hours, the mixture was cured at 37°C for 20 minutes, coating the intermediate layer with the outer layer to obtain a biotin-cross-linked HA sustained-release formulation.

[0082] Experimental methods and materials:

[0083] Cell toluidine blue staining: After fixing the cells with 4% paraformaldehyde for 10 min, stain them with 0.1% toluidine blue staining solution (pH 4.0) at room temperature for 10 min, and then wash them with PBS until they no longer change color.

[0084] Primary chondrocyte extraction from mice: 7-10 suckling mice within 3 days of birth were anesthetized with tribromoethanol at a standard concentration of 10 μl / g, then euthanized by cervical indwelling. The mice were then disinfected in 75% alcohol for 15 minutes and transferred to sterile dishes. The knee joint was separated using autoclaved tissue scissors and forceps. The skin, ligaments, muscles, and fat around the knee joint were carefully removed, and the cartilage caps at the tips of the tibia and femur were carefully peeled off and placed in sterile Eppendorf tubes. 0.25% trypsin was added, and the mixture was incubated at 37°C for 30 minutes. Subsequently, microtweezers were used twice to thoroughly remove any fascia and fibers adhering to the surface of the cartilage block. The cleaned cartilage block was placed in centrifuge tubes containing 0.1% type II collagenase and 10% fetal bovine serum (FBS) in DMEM / F12 complete medium and digested at 37°C for 12 hours. When the cartilage blocks are digested to the point where they are no longer visible to the naked eye, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the chondrocytes in DMEM / F12 complete medium containing penicillin-streptomycin antibiotic 100X and 10% fetal bovine serum (FBS), and then seed them into 10 cm cell culture dishes (this is generation P0) and culture at 37°C with 5% CO2.

[0085] Osteoarthritis cell model construction: According to previous studies, mouse or human chondrocytes can be successfully constructed by stimulating them with IL-1β (the inflammatory factor interleukin-1β) at a final concentration of 10 ng / ml for 48 hours.

[0086] Western blotting (WB): Primary mouse chondrocytes were extracted and cultured. After treatment with IL-1β and biotin, cellular proteins were extracted from the treated group, control group, and blank control group for Western blotting analysis. Cellular proteins were extracted on ice using Ripa cell lysis buffer and protease inhibitors. The lysates were then separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane by wet transfer. Next, the PVDF membrane was blocked with 5% skim milk on a decolorizing slow shaker at room temperature for 1 hour, followed by incubation with primary antibody at 4°C for 12–16 hours. The next day, the PVDF membrane was incubated with secondary antibody at room temperature for 1 hour. Finally, protein band images were acquired using a chemiluminescence analyzer (Tanon, model 5200CE) with sensitive ECL luminescence solution.

[0087] C57BL / 6J mice were purchased from Jiangsu Gempharmatech Co., Ltd., strain: C57BL / 6JGpt, serial number: N000013. Mice were housed in a specific pathogen-free (SPF) animal room, maintaining a temperature of 20-26℃, humidity of 50%-60%, and a 12-hour light / dark cycle. The experimental mice were fed a pelleted maintenance diet (1022) purchased from Beijing Huafukang Biotechnology Co., Ltd., batch number 2310220724, standard: GB 14924-2010. All food and water were autoclaved before feeding the mice.

[0088] Animal Model of Osteoarthritis (DMM-OA): DMM-OA is an osteoarthritis model induced by medial meniscus instability. Ten-week-old C57BL / 6J wild-type mice or SD rats were selected. The right posterior knee joint was operated on: After anesthesia, the mice were prepared and disinfected. The skin on the medial side of the joint was incised, and the patellar ligament was dissected along the medial side of the knee joint to expose the joint cavity. The medial meniscus and tibial ligament were severed, and the medial meniscus was freed to induce knee instability. After completion, the joint cavity was closed, and the skin was sutured. Sham-operated group mice: The skin of the right knee joint was simply incised and sutured. Knee joint specimens were collected 8 weeks post-operation. The severity of OA was assessed using the OARSI (Osteoarthritis Research Society International) scoring system (0-6 points).

[0089] Oral administration of drugs to mouse knee joints: Biotin was dissolved in drinking water to obtain low-concentration biotin (L-Vit B7) and high-concentration biotin (H-Vit B7). Mice ingested the drug by drinking water, and the drinking water was changed every 2 days to ensure the effectiveness of the drug. Mice with OA induced by DMM model were treated with the drug by continuously administering the drug through drinking water for 8 weeks.

[0090] Drug injection into the rat knee joint: After anesthetizing the rat, clean the slightly medial aspect of the bent knee joint with a 70% alcohol swab. Using a 1mL insulin syringe containing the drug, after confirming that the needle has entered the abdominal cavity (a sudden decrease in resistance may be felt), slowly inject the drug. Inject the biotin sustained-release formulation prepared in the above example, slowly withdraw the needle, and observe the injection site for any abnormal bleeding or drug leakage. Return the rat to its cage and continue to observe for several minutes to ensure no adverse reactions occur.

[0091] Human knee cartilage explant culture: After obtaining informed consent, joint tissue was collected from OA patients undergoing knee replacement surgery at our hospital. Patients with malignant tumors, diabetes, or other serious chronic diseases within the past five years were excluded to ensure that all OA specimens selected in this study were caused by degenerative osteoarthritis. Freshly harvested OA cartilage specimens from OA patients undergoing total knee replacement surgery were cut into approximately 1cm pieces. 3 The cartilage specimens were washed three times with PBS buffer containing 1% penicillin and streptomycin. Immediately afterward, the cartilage blocks were transferred to 12-well plates, and 1.5 ml of DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added. To assess the therapeutic effect of the drug on OA cartilage, half of the cartilage specimens in one well of the 12-well plate were incubated with biotin, while the remaining half was incubated with dimethyl sulfoxide as a control. The 12-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 14 days, with medium changes and drug additions every other day. After 14 days of culture, the specimens were fixed with 4% paraformaldehyde, dehydrated, embedded, and sectioned.

[0092] Gross wear score in rats: The Pelletier score assesses the surface condition, color, fissures, and depth of articular cartilage damage, classifying the degree of damage into five levels: 0 (smooth articular surface, normal color) to 4 (cartilage detachment, subchondral bone exposure). 0: Smooth articular surface, normal color, smooth joint surface. 1: Rough articular surface, small fissures, and dark color. 2: Bone defect reaching the middle layer of cartilage. 3: Ulceration of the cartilage surface, cartilage defect reaching the deep layer of cartilage. 4: Cartilage detachment, exposing subchondral bone.

[0093] Safranin O-Rapid Green Staining: Dewaxed and hydrated tissue sections were immersed in PBS for 5 minutes, stained with a prepared 1% Fast Green solution for 60 seconds, fixed with 3% acetic acid fixative for 3 seconds, and then washed off. Next, they were stained with 0.5% Safranin O solution for 30 seconds, and washed with deionized water to remove any excess stain. Finally, after dehydration and clearing, they were sealed with neutral resin. Images were acquired using an Olympus upright microscope, and three experienced researchers blindly scored the severity of articular cartilage osteoarthritis (OA) using the OARSI (Osteoarthritis Research Society International) grading system (0-6 points).

[0094] Immunofluorescence (IF): Mouse chondrocyte line ATDC5, primary mouse chondrocytes, and human chondrocyte line C28 / I2 were seeded in confocal dishes. After the cells reached a suitable density, appropriate stimulation was added for 48 hours. Cells were then fixed with 4% paraformaldehyde for 20-30 minutes, followed by washing three times with PBS. Next, permeabilization was performed with 0.1%-0.2% Triton X-100 for 10 minutes, followed by washing again with PBS. Afterward, blocking was performed with blocking buffer (e.g., 10% FBS or 5% BSA) at room temperature for 30 minutes to reduce non-specific binding. Then, diluted primary antibody was added to the sample and incubated at room temperature for 1 hour or overnight at 4°C, followed by washing three times with PBS. Next, secondary antibody of the same species as the primary antibody was added and incubated at room temperature for 1 hour or at 37°C for 1.5 hours, followed by washing three times with PBS. If necessary, the cell nuclei could be stained with DAPI, stained at room temperature for 10 minutes, followed by washing three times with PBS. Finally, the slides are mounted with anti-fluorescence quenching mounting medium, and then observed and images are acquired under a fluorescence microscope or a laser confocal microscope.

[0095] Main experimental reagents:

[0096] DMEM / F12 basal medium (Gibco; catalog number: C11330500BT). Australian fetal bovine serum (Gibco; catalog number: 10099-141C). Penicillin-streptomycin antibiotic 100X solution (Kangning; catalog number: 30-002-CI). 0.25% Trypsin-EDTA (Gibco; catalog number: 25200-072). Collagenase type II (Sigma-Aldrich; catalog number: C-BIOC). Bone tissue Safranine O and Fast Green staining solution (Sigma Aldrich). Protease inhibitor (Thermo Fisher; catalog number: 87785), Ripa cell lysis buffer (Thermo Fisher; catalog number: 89901), protein marker (Thermo Fisher; catalog number: 26616). 30% polyacrylamide-methylenebisacrylamide / Acr-Bis (Beijing Regen Biotech). Skim milk powder (Inner Mongolia Yili). Sensitive ECL chemiluminescence solution (MedChemExress; catalog number HY-K1005). PVDF membrane (Sigma Aldrich; catalog number ISEQ00010). 0.3% hydrogen peroxide (Guangzhou Sanxun). 4% paraformaldehyde (Beijing Lanjieke). Neutral resin, goat serum (Guangzhou Solarbio). DMSO (MedChemExress). DAPI (Thermo Fisher; D1306). Antibodies are listed in Table 1.

[0097] Table 1

[0098]

[0099] Results of Western blot analysis of cellular proteins and toluidine blue staining are as follows: Figure 1 As shown, the results indicate that through WB ( Figure 1 A) and toluidine blue staining ( Figure 1 (B) The study examined the chondrogenic differentiation of mesenchymal stem cells under stimulation with different concentrations of biotin (B7). Compared with the control group and the insulin-transferrin-selenium triple additive (ITS), biotin promoted the expression of SOX9 and COL2. Toluidine blue staining also showed that biotin could promote the synthesis of cartilage matrix and promote the chondrogenic differentiation of mesenchymal stem cells.

[0100] Cell immunofluorescence assay results as follows Figure 2 As shown, the results indicate that by analyzing ATDC5 ( Figure 2 A), C28 / I2 ( Figure 2 B) and mouse chondrocytes ( Figure 2In the study C), after adding IL-1β to stimulate an in vitro arthritis model, cells were stained with COL2 fluorescence. It was found that Biotin treatment could significantly alleviate the degradation of COL2, a chondrocyte synthesis marker induced by IL-1β, indicating that Biotin can counteract the effect of IL-1β on chondrocyte matrix degradation and may serve as a potential therapeutic drug for OA.

[0101] Results of oral drug treatment of mouse knee joints Figure 3 As shown, the results indicate that high concentrations of biotin can significantly improve OARSI scores for arthritis and slow the progression of arthritis.

[0102] Results of human knee cartilage explant culture Figure 4 As shown, the results indicated that after 14 days of in vitro culture of lesioned cartilage specimens from OA patients undergoing total knee arthroplasty via biotin, biotin significantly inhibited the degradation of chondroitin proteoglycans and fibrosis, suggesting that biotin has clinical therapeutic potential.

[0103] Results of drug injection into the knee joint of rats as follows Figure 5 As shown, the results indicate that the use of biotin sustained-release formulations in rat arthritis... Figure 5 In the figure, A represents the in vitro drug release curve, showing that the drug release rate of biotin within 15 minutes is 60.33%. Figure 5 B in the diagram shows that intra-articular injection of the biotin sustained-release formulation significantly reduced the degree of joint surface wear. Figure 5 The C in the chart shows that the Pelletier score has declined.

[0104] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. Use of biotin as an active ingredient in the preparation of a drug for slowing the progression of osteoarthritis.

2. Use of biotin as an active ingredient in the preparation of a drug for treating and / or preventing osteoarthritis.

3. Use of a biotin sustained-release preparation as an active ingredient in the preparation of a drug for slowing the progression of osteoarthritis.

4. Use of a biotin sustained-release preparation as an active ingredient in the preparation of a drug for treating and / or preventing osteoarthritis.

5. Use according to claim 3 or 4, characterized in that, The biotin sustained-release preparation comprises a sustained-release particle, an intermediate layer and an outer layer; the sustained-release particle comprises a sustained-release material and biotin, the intermediate layer coats the sustained-release particle, the intermediate layer comprises a cationic liposome, and the outer layer coats the intermediate layer, the outer layer comprises a cross-linked hyaluronic acid.

6. Use according to claim 5, characterized in that, The particle size of the sustained-release particle is 200 nm ± 50 nm.

7. Use according to claim 5, characterized in that, At least one of the following features is included: (1) The sustained-release material comprises a high molecular weight sustained-release material or a small molecular weight sustained-release material, the high molecular weight sustained-release material is selected from one or more of sodium alginate, chitosan, polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyric acid, polyhydroxybutyric acid-hydroxyvaleric acid copolymer, polyortho ester, polyanhydride and cross-linked hyaluronic acid; and the small molecular weight sustained-release material is selected from a lipid compound; (2) The sustained-release particle further comprises an emulsifier.

8. Use according to claim 7, characterized in that, At least one of the following features is included: (1) The sustained-release material comprises a high molecular weight sustained-release material, and at least one of the following conditions is satisfied: (a) the weight average molecular weight of the high molecular weight sustained-release material is 10,000 Da to 100,000 Da; and (b) the mass ratio of biotin to high molecular weight sustained-release material is (10-50):(0.1-2); (2) The emulsifier is selected from one or more of polyvinyl alcohol, Tween 80, PEG-100 stearate, glyceryl stearate, arachidyl alcohol, fatty alcohol polyoxyethylene ether, glyceryl behenate, cetyl stearyl glucoside and poloxamer 407; (3) The added mass of the emulsifier accounts for 3% to 7% of the mass of the sustained-release material.

9. Use according to any one of claims 6 to 8, characterized in that, At least one of the following features is included: (1) The cationic liposome comprises (2,3-dioleoyl-propyl)-trimethylamine; (2) The intermediate layer further comprises a zinc ion-polyphenol complex; (3) The mass ratio of the intermediate layer to the sustained-release particle is 1:(2.5-4.5); (4) The cross-linking agent of the cross-linked hyaluronic acid comprises divinyl sulfone; (5) The cross-linking degree of the cross-linked hyaluronic acid is 15% to 25%; (6) The weight average molecular weight of the cross-linked hyaluronic acid is 1.0 MDa to 1.5 MDa; (7) The mass ratio of the outer layer to the sustained-release particle is 1:(1.3-1.8).

10. Use according to claim 3 or 4, characterized in that, The preparation method of the biotin sustained-release preparation comprises the following steps: Preparation of a sustained-release particle, the sustained-release particle comprising a sustained-release material and biotin; Coating of an intermediate layer on the sustained-release particle, the intermediate layer comprising a cationic liposome; Coating of an outer layer on the intermediate layer, the outer layer comprising a cross-linked hyaluronic acid.

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