Trichosanthin sustained-release injection and its preparation method and application
By using intra-articular injection of a composite formulation of PLGA nanoparticles and hyaluronic acid hydrogel, the problems of low bioavailability and systemic toxicity of tripterygium wilfordii have been solved, achieving localized long-term sustained release and safe treatment of rheumatoid arthritis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING MEDICAL UNIVERSITY
- Filing Date
- 2026-04-30
- Publication Date
- 2026-06-02
Smart Images
Figure CN122123970A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a tripterygium acid sustained-release injection, its preparation method, and its application. Background Technology
[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by synovial hyperplasia, progressive destruction of articular cartilage and bone, ultimately leading to loss of joint function. The global prevalence of RA is currently 0.5%–1%, with approximately 5 million patients in China. Clinical treatments for RA include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, traditional disease-modifying antirheumatic drugs (DMARDs), and biologics. However, these drugs generally suffer from low bioavailability, require frequent administration, and have poor targeting; long-term use can cause adverse reactions such as nausea, abdominal pain, gastric ulcers, and osteoporosis.
[0003] Tripterygium wilfordii Hook.f. is a traditional Chinese medicine used to treat rheumatoid arthritis (RA), possessing properties such as dispelling wind and dampness, promoting blood circulation, reducing swelling and relieving pain. Modern pharmacological studies have shown that the alkaloids, diterpenes, and triterpenes in Tripterygium wilfordii have immunosuppressive and anti-inflammatory effects, inhibiting the proliferation of inflammatory cells and the release of cytokines. However, Tripterygium wilfordii extract has a complex composition, with some components exhibiting strong toxicity. Oral administration results in low local drug concentrations in the joints, and long-term use increases the risk of gastrointestinal, hepatic, and renal damage. Therefore, reducing the toxicity of the active components of Tripterygium wilfordii and improving its bioavailability is a key challenge in developing Tripterygium wilfordii-based RA treatments.
[0004] Direct injection of drugs into the affected joint cavity enables targeted local drug delivery, significantly increasing intra-articular drug concentration and reducing systemic exposure. Polylactic acid-glycolic acid copolymer microspheres / nanoparticles are a biodegradable and highly safe sustained-release carrier that can prolong the duration of drug action locally. Hyaluronic acid hydrogels possess good biocompatibility, injectability, and controlled drug release capabilities, and can serve as a dispersion medium for microspheres / nanoparticles, forming a dual sustained-release system. Currently, there are no reports on the preparation of tripterygium acid into a PLGA nanoparticle-hyaluronic acid hydrogel composite formulation for intra-articular injection therapy of rheumatoid arthritis (RA). Summary of the Invention
[0005] The purpose of this invention is to provide a triptolide sustained-release injection, its preparation method, and its application, thereby solving the technical problems of poor water solubility, low oral bioavailability, and significant systemic toxicity of existing triptolide, achieving long-term local sustained release in the joint cavity, and improving the treatment efficacy and safety of rheumatoid arthritis (RA).
[0006] In view of this, one of the objectives of the present invention is to provide a tripterygium acid sustained-release injection, which is prepared by comprising the following steps:
[0007] 1) Weigh 20 mg of polylactic acid-glycolic acid copolymer and dissolve it in 1 mL of dichloromethane to obtain an oil phase; the molar ratio of lactic acid to glycolic acid units in the polylactic acid-glycolic acid copolymer is 75:25, and the polylactic acid-glycolic acid copolymer is ester-terminated.
[0008] 2) Weigh 2 mg of tripterygic acid and dissolve it in 400 μL of an aqueous solution containing 0.2% formic acid and 8% acetonitrile to obtain the inner aqueous phase;
[0009] 3) Under the conditions of ultrasonic power of 100 W and ultrasonication for 5 s followed by 3 s pause, the inner aqueous phase was slowly added dropwise to the oil phase for 3 minutes to obtain the primary emulsion;
[0010] 4) Add the colostrum to 10 mL of a 4% (w / v) polyvinyl alcohol aqueous solution and emulsify for 4 min under ultrasonic power of 300 W and ultrasonication for 5 s followed by 3 s pause to obtain a secondary emulsion;
[0011] 5) Stir magnetically for 3 h to evaporate dichloromethane, centrifuge, wash, and freeze-dry to obtain nanoparticle powder;
[0012] 6) Weigh 25 mg of sodium hyaluronate powder, add 1 mL of triple-distilled water, and vortex to dissolve, to obtain 2.5% hyaluronic acid hydrogel;
[0013] 7) Mix 5 mg of nanoparticle powder with 1 mL of hyaluronic acid hydrogel to obtain tripterygium acid sustained-release injection.
[0014] A second objective of this invention is to provide a method for preparing a tripterygium hydrate sustained-release injection, comprising the following steps:
[0015] 1) Weigh 20-100 mg of polylactic acid-glycolic acid copolymer and dissolve it in 1 mL of dichloromethane to obtain an oil phase; the molar ratio of lactic acid to glycolic acid units in the polylactic acid-glycolic acid copolymer is 75:25, and the polylactic acid-glycolic acid copolymer is ester-terminated.
[0016] 2) Weigh 2 mg of tripterygic acid and dissolve it in 400 μL of an aqueous solution containing 0.2% formic acid and 8% acetonitrile to obtain the inner aqueous phase;
[0017] 3) Under the conditions of ultrasonic power of 100 W and ultrasonication for 5 s followed by 3 s pause, the inner aqueous phase was slowly added dropwise to the oil phase for 3 minutes to obtain the primary emulsion;
[0018] 4) Add the colostrum to 10 mL of a 1%~4% polyvinyl alcohol aqueous solution and emulsify for 4 min under ultrasonic power of 300W, ultrasonic 5 s / 3 s pause, to obtain a secondary emulsion;
[0019] 5) Stir magnetically for 3 h to evaporate dichloromethane, centrifuge, wash, and freeze-dry to obtain nanoparticle powder;
[0020] 6) Weigh 25 mg of sodium hyaluronate powder, add 1 mL of triple-distilled water, and vortex to dissolve, to obtain 2.5% hyaluronic acid hydrogel;
[0021] 7) Mix 5 mg of nanoparticle powder with 1 mL of hyaluronic acid hydrogel to obtain tripterygium acid sustained-release injection.
[0022] Preferably, the polylactic acid-glycolic acid copolymer is 20 mg; and the polyvinyl alcohol aqueous solution has a mass-volume concentration of 4%.
[0023] A third objective of this invention is to provide the application of the triptolide sustained-release injection or the triptolide sustained-release injection obtained by the above-described preparation method in the preparation of drugs for treating rheumatoid arthritis.
[0024] The fourth objective of this invention is to provide a medicament for treating rheumatoid arthritis, comprising the tripterygium acid sustained-release injection described above or the tripterygium acid sustained-release injection prepared by the method described above.
[0025] Furthermore, the dosage form of the drug includes solutions, capsules, pills, granules, sprays, or injections.
[0026] Furthermore, the drug contains one or more pharmaceutically acceptable carriers or excipients.
[0027] The fifth objective of this invention is to provide a method for constructing a collagen-induced arthritis mouse model, comprising the steps of taking female DBA / 1 mice, subcutaneously injecting 100 μL of emulsion into the tail root, and injecting emulsion again 21 days later to enhance immunization.
[0028] The model was considered successful when mice showed redness and swelling of the toes between days 28 and 35. The emulsion was obtained by mixing bovine type II collagen and complete Freund's adjuvant in a 1:1 ratio.
[0029] This invention prepares tripterygic acid (PLGA) nanoparticles via a double emulsion-solvent evaporation method. The optimal formulation was determined by optimizing the amount of PLGA (polylactic acid-glycolic acid copolymer) and the concentration of PVA (polyvinyl alcohol): 20 mg PLGA and 4% PVA (w / v). The resulting nanoparticles had a particle size of approximately 400.0 ± 112.8 nm, an encapsulation efficiency of approximately 60%, and a drug loading of approximately 1.16%.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] 1. Reduced systemic toxicity: By injecting locally into the joint cavity, the drug mainly remains in the joint cavity, reducing systemic exposure and avoiding liver and kidney damage and gastrointestinal reactions caused by oral Tripterygium wilfordii preparations.
[0032] 2. Dual sustained-release effect: PLGA (polylactic acid-glycolic acid copolymer) nanoparticles provide the first level of sustained release, and hyaluronic acid hydrogel provides the second level of sustained release. The drug can be continuously released in the joint cavity for several days to several weeks, reducing the frequency of administration.
[0033] 3. CIA mouse model: The method for constructing the CIA mouse model of this invention is simple and quick, and can accurately simulate the pathological characteristics of patients with rheumatoid arthritis.
[0034] 4. Significant anti-inflammatory effect: In the CIA (collagen-induced arthritis) mouse model (the collagen-induced arthritis model is currently the most widely used animal model of RA), this preparation significantly reduced toe swelling, improved knee cartilage damage and synovial hyperplasia, reduced serum levels of inflammatory factors such as IL-1β, TNF-α, IL-4, and IL-10, and increased IL-6 levels to restore immune function.
[0035] 5. Protects against organ damage: This preparation can reduce splenomegaly in CIA model mice, improve histopathological damage to the liver and kidneys, and serum ALT, AST and other indicators show hepatoprotective effects.
[0036] 6. Controllable preparation process: The double emulsion-solvent evaporation method is simple to operate, the nanoparticles are uniform in size, the hydrogel has good injectability, and it is easy to translate into clinical applications. Attached Figure Description
[0037] Figure 1 The images show the high-performance liquid chromatograms and method validation results for triptolide. In the figures, a is the chromatogram of triptolide reference standard, b is the chromatogram of blank PLGA nanoparticles, c is the chromatogram of PVA, and d is the fingerprint chromatogram.
[0038] Figure 2 Characterization results of PLGA (Tripterygium wilfordii) nanoparticles. In the figures, a and b show the nanoparticle solution appearance, c shows the particle size distribution, d shows the Zeta potential, and e and f show transmission electron microscopy (TEM) images.
[0039] Figure 3 The following describes the characteristics of hyaluronic acid hydrogel. Here, a represents the appearance of the hydrogel, and b represents the injectability of the hydrogel (which may be labeled "RA" or "ZG").
[0040] Figure 4 The therapeutic effect of triptolide sustained-release injection on CIA mice is shown. In the figures, a) is a representative photograph of mouse paw swelling, b) is the change in body weight during treatment, c) is the paw swelling curve, d) is the spleen index, and e) is the liver index.
[0041] Figure 5 The effects of triptolide sustained-release injection on the internal organs of CIA mice are shown. A, B, C, and D represent serum biochemical indicators (AST, ALT, CRE, UA), and e represents the H&E staining results of the heart, liver, spleen, and kidney.
[0042] Figure 6 This study evaluates the in vivo anti-inflammatory and joint-protective effects of triptolide sustained-release injection. A, b, c, d, and e represent serum inflammatory factor levels (IL-1β, IL-4, IL-10, IL-6, TNF-α), and f represents the results of H&E staining and Safranin O-Fixed Green staining of the knee joint. Detailed Implementation
[0043] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are all conventional commercially available products.
[0044] Example 1: Establishment of a method for determining tripterygic acid content
[0045] The content of triptolide was determined by high performance liquid chromatography (HPLC). Chromatographic conditions: Wondasil C18 column (4.6 × 250 mm, 5 μm); mobile phase: methanol-0.2% formic acid aqueous solution (92:8, v / v); flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 262 nm; injection volume: 10 μL.
[0046] Specificity test: The chromatographic peak shape of tripterygic acid reference standard was good, and there was no interference from blank excipients at the retention time.
[0047] Precision test: The intraday RSD for low, medium, and high concentrations was less than 2%.
[0048] Stability test: The peak area RSD within 24 h was 0.57%.
[0049] Repeatability test: RSD was 1.47% for 6 measurements.
[0050] Linearity: Within the concentration range of 40–200 μg / mL, the peak area showed a good linear relationship with the concentration (R²>0.999).
[0051] The method meets the requirements for specificity, precision, stability, and repeatability, and can be used for the determination of tripterygic acid content. Figure 1 ).
[0052] Example 2: Preparation and optimization of tripterygic acid PLGA nanoparticles
[0053] The double emulsion-solvent evaporation method (W / O / W) was adopted.
[0054] (1) Oil phase: Weigh 20 mg of PLGA (polylactic acid-glycolic acid copolymer, LA:GA=75:25, ester-terminated) and dissolve it in 1 mL of dichloromethane.
[0055] (2) Inner aqueous phase: Weigh 2 mg of tripterygium acid and dissolve it in 400 μL of aqueous solution (containing 0.2% formic acid and 8% acetonitrile).
[0056] (3) Colostrum: Under ultrasonic conditions (ultrasonic power 100 W, ultrasonic 5 s / stop 3 s), the inner aqueous phase is slowly added dropwise to the oil phase for 3 min.
[0057] (4) Re-emulsification: Add the colostrum to a 4% PVA aqueous solution (10 mL) and emulsify for 4 min under ultrasonic conditions (ultrasonic power 300 W, ultrasonic 5 s / stop 3 s).
[0058] (5) Curing: Stir magnetically for 3 h to evaporate dichloromethane, centrifuge (10000 rpm, 4℃, 20 min), wash three times with deionized water, freeze dry to obtain nanoparticle powder.
[0059] The optimization results show that the nanoparticles prepared with different PLGA dosages are shown in Table 1; the nanoparticles prepared with different PVA concentrations are shown in Table 2. When the PLGA dosage is 20 mg and the PVA (polyvinyl alcohol) concentration is 4%, the amount of material used is the least, the encapsulation efficiency is the highest (EE), reaching 60.58±0.91%, and the drug loading is 1.16±0.13%. Blank nanoparticles were prepared using the same method (without triptolic acid).
[0060] Table 1 Nanoparticles prepared with different PLGA dosages
[0061]
[0062] Table 2. Nanoparticles prepared with different PVA concentrations
[0063]
[0064] Example 3 Characterization of PLGA tripterygic acid nanoparticles
[0065] (1) Particle size and Zeta potential: The nanoparticles were diluted with ultrapure water at a mass-to-volume ratio of 1 mg:25 mL and measured using a dynamic light scattering instrument. The results are as follows: Figure 2As shown in a~d, the average particle size is 400.0±112.8 nm, the polydispersity index is 0.144±0.054, and the zeta potential is -8.77±0.22 mV.
[0066] (2) Morphological observation: Transmission electron microscopy showed that the nanoparticles were spherical, with smooth surfaces, no aggregation, and uniform particle size distribution. Figure 2 e~f).
[0067] Example 4: Preparation and Injectability Evaluation of Hyaluronic Acid Hydrogel
[0068] Weigh 25 mg of sodium hyaluronate powder (molecular weight 200,000-400,000), add 1 mL of triple-distilled water, and vortex to dissolve, thus obtaining 2.5% hyaluronic acid hydrogel. To observe injectability, a small amount of methylene blue was added for staining. The hydrogel was drawn into a 1 mL syringe and squeezed out through the needle. The results are as follows. Figure 3 As shown, the hydrogel can be continuously extruded and can accurately write the letters "RA" and "ZG" with uniform and continuous lines, indicating good injectability.
[0069] Example 5: Compound of Tripterygium wilfordii acid sustained-release injection
[0070] Weigh 5 mg of the freeze-dried nanoparticle powder prepared in Example 2, add 1 mL of the hyaluronic acid hydrogel prepared in Example 4, and gently vortex to disperse the nanoparticles evenly, thus obtaining the tripterygium acid sustained-release injection.
[0071] Example 6: In vivo pharmacodynamic evaluation
[0072] (1) Establishment of CIA (collagen-induced arthritis) mouse model: Female DBA / 1 mice were injected subcutaneously into the base of the tail with 100 μL of an emulsion (a 1:1 mixture of bovine type II collagen and complete Freund's adjuvant). The emulsion was injected again 21 days later to boost immunity. The model was considered successful when the mice developed paw swelling and redness between days 28 and 35. Figure 4 a).
[0073] (2) Grouping and administration: The model mice were randomly divided into a model group, a low-dose triptolide group (L, 0.5 mg / kg), a medium-dose group (M, 1.0 mg / kg), and a high-dose group (G, 1.5 mg / kg), with 6 mice in each group. A normal control group was also set up. Each group was administered the medication via intra-articular injection once every 4 days until the end of day 60.
[0074] (3) Measurement of toe swelling: Toe thickness was measured every 4 days. The results showed that the toe thickness in the model group was significantly higher than that in the normal group (P<0.05); the toe thickness in each treatment group was significantly lower than that in the model group and was close to that in normal mice (P<0.05), with the medium-dose group showing the best effect (2.03±0.06 mm vs. 2.83±0.31 mm in the model group). Figure 4 c).
[0075] (4) Body weight and organ index: There was no significant difference in body weight among the groups during the drug administration period. Figure 4 b). The spleen index was significantly increased in the model group (P<0.001), while it was significantly decreased in the low- and high-dose groups (P<0.05). Figure 4 d, e).
[0076] (5) Serum biochemical indicators: AST and ALT were abnormally elevated in the model group; ALT was significantly decreased in all treatment groups (P<0.001), while AST returned to normal levels. Figure 5 )
[0077] (6) Detection of inflammatory factors: ELISA results showed that the levels of IL-1β, TNF-α, IL-4, and IL-10 were significantly increased in the model group (P<0.001); the levels of IL-1β, TNF-α, IL-4, and IL-10 were significantly decreased in all treatment groups (P<0.05), while the level of IL-6 significantly increased (P<0.001). Figure 6 a~e)
[0078] (7) Histopathology: H&E and Safranin O-Fixed Green staining of the knee joint showed severe cartilage destruction and inflammatory cell infiltration in the model group; the cartilage structure of all treatment groups, especially the medium and high dose groups, was significantly improved, and the inflammatory infiltration was reduced. H&E staining of major organs showed local lesions in the liver, spleen, and kidneys of the model group; the degree of lesions was reduced in all treatment groups, with the high dose group showing the most significant improvement. Figure 5 e Figure 6 f)
[0079] All experimental data in this invention are expressed as mean ± standard deviation. A t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.
[0080] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A sustained-release injection of tripterygium wilfordii acid, characterized in that, It is prepared by the following steps: 1) Weigh 20 mg of polylactic acid-glycolic acid copolymer and dissolve it in 1 mL of dichloromethane to obtain an oil phase; the molar ratio of lactic acid to glycolic acid units in the polylactic acid-glycolic acid copolymer is 75:25, and the polylactic acid-glycolic acid copolymer is ester-terminated. 2) Weigh 2 mg of tripterygic acid and dissolve it in 400 μL of an aqueous solution containing 0.2% formic acid and 8% acetonitrile to obtain the inner aqueous phase; 3) Under the conditions of ultrasonic power of 100 W and ultrasonication for 5 s followed by 3 s pause, the inner aqueous phase was slowly added dropwise to the oil phase for 3 min to obtain the primary emulsion; 4) Add the colostrum to 10 mL of a 4% (w / v) polyvinyl alcohol aqueous solution and emulsify for 4 min under ultrasonic power of 300 W and ultrasonication for 5 s followed by 3 s pause to obtain a secondary emulsion; 5) Stir magnetically for 3 h to evaporate dichloromethane, centrifuge, wash, and freeze-dry to obtain nanoparticle powder; 6) Weigh 25 mg of sodium hyaluronate powder, add 1 mL of triple-distilled water, and vortex to dissolve, to obtain 2.5% hyaluronic acid hydrogel; 7) Mix 5 mg of nanoparticle powder with 1 mL of hyaluronic acid hydrogel to obtain tripterygium acid sustained-release injection.
2. A method for preparing a tripterygium acid sustained-release injection, characterized in that, Includes the following steps: 1) Weigh 20-100 mg of polylactic acid-glycolic acid copolymer and dissolve it in 1 mL of dichloromethane to obtain an oil phase; the molar ratio of lactic acid to glycolic acid units in the polylactic acid-glycolic acid copolymer is 75:25, and the polylactic acid-glycolic acid copolymer is ester-terminated. 2) Weigh 2 mg of tripterygic acid and dissolve it in 400 μL of an aqueous solution containing 0.2% formic acid and 8% acetonitrile to obtain the inner aqueous phase; 3) Under the conditions of ultrasonic power of 100 W and ultrasonication for 5 s followed by 3 s pause, the inner aqueous phase was slowly added dropwise to the oil phase for 3 min to obtain the primary emulsion; 4) Add the colostrum to 10 mL of a 1%~4% polyvinyl alcohol aqueous solution and emulsify for 4 min under ultrasonic power of 300 W and ultrasonication for 5 s followed by 3 s pause to obtain a secondary emulsion; 5) Stir magnetically for 3 h to evaporate dichloromethane, centrifuge, wash, and freeze-dry to obtain nanoparticle powder; 6) Weigh 25 mg of sodium hyaluronate powder, add 1 mL of triple-distilled water, and vortex to dissolve, to obtain 2.5% hyaluronic acid hydrogel; 7) Mix 5 mg of nanoparticle powder with 1 mL of hyaluronic acid hydrogel to obtain tripterygium acid sustained-release injection.
3. The method as described in claim 2, characterized in that, The polylactic acid-glycolic acid copolymer is 20 mg; the polyvinyl alcohol aqueous solution has a mass-volume concentration of 4%.
4. The use of the triptolide sustained-release injection according to claim 1 or the triptolide sustained-release injection obtained by the preparation method according to claim 2 in the preparation of drugs for treating rheumatoid arthritis.
5. A drug for treating rheumatoid arthritis, characterized in that, The product comprises the triptolide sustained-release injection according to claim 1 or the triptolide sustained-release injection obtained by the preparation method according to claim 2.
6. The drug as described in claim 5, characterized in that, The dosage forms of the drug include solutions, capsules, pills, granules, sprays, or injections.
7. The drug as described in claim 5, characterized in that, The drug contains one or more pharmaceutically acceptable carriers or excipients.
8. A method for constructing a mouse model of collagen-induced arthritis, characterized in that, The procedure includes taking female DBA / 1 mice, subcutaneously injecting 100 μL of emulsion into the base of the tail, and then injecting the emulsion again 21 days later to boost immunity. The model was considered successful when mice showed redness and swelling of the toes between days 28 and 35. The emulsion was obtained by mixing bovine type II collagen and complete Freund's adjuvant in a 1:1 ratio.