Triptolide wood wax acid ester and its liposome and preparation method thereof

Abstract

The application relates to the technical field of medicines, in particular to a triptolide wood wax acid ester, a liposome thereof and a preparation method, a chemical structural formula of the triptolide wood wax acid ester is shown as formula (I): the triptolide wood wax acid ester provided by the application is obtained by esterification of triptolide C14-OH and wood wax acid, the effectiveness, safety and the like of the triptolide wood wax acid ester liposome against tumors are further improved compared with the prior art, so that a foundation is laid for providing a safer and more effective triptolide related preparation for clinic.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a triptolide oxyphylla ester, its liposomes, and a preparation method thereof. Background Technology

[0002] Triptolide (TP), also known as triptolide or triptolide alcohol, is an epoxy diterpenoid lactone compound isolated from the Chinese medicinal plant Tripterygium wilfordii Hook F. It has a wide range of pharmacological effects, including anti-inflammatory, immunosuppressive, antitumor, and antifertility effects (Liu Q. Triptolide and its expanding multiple pharmacological functions[J]. International Immunopharmacology, 2011, 11(3):377-383). In the field of oncology, TP has shown effective antitumor activity in various cancers, and its antitumor effects have been extensively studied. TP has shown good antitumor activity in nearly 60 tumor cell lines (NOEL PVONHOFF DD, SALUJA AK, et al. Triptolide and its derivatives as cancer therapies[J]. Trends Pharmacol Sci, 2019, 40(5):327-341), exhibiting broad-spectrum and high-efficiency characteristics.

[0003] However, the effective dose of TP is close to its toxic dose, resulting in a narrow therapeutic window (Song Yi, Liu Yan, Fang Bingqian, Kang Di, Hu Lihong. Research progress on the antitumor effect of triptolide and its compatibility to reduce toxicity [J]. Journal of Nanjing University of Traditional Chinese Medicine, 2021, 37(03):457-464.). It also suffers from a short half-life and poor formulation properties, severely limiting its clinical application. Therefore, no highly effective drug targeting TP for antitumor treatment has been developed and marketed to date. How to prolong the in vivo action time and reduce toxic side effects while maintaining the biological activity of TP is a key issue that urgently needs to be addressed to ensure its safe and effective clinical application.

[0004] Prodrugs are new compounds formed by chemical bonds between a drug and a modifying group, which can be enzymatically hydrolyzed by an in vivo enzyme system to exert their pharmacological effects. Prodrug design is a common method for antitumor drugs to change their physicochemical properties. The main purpose is to improve the drug's digestion, absorption, distribution, and metabolism (ADME), prolong its half-life, increase its bioavailability, increase its stability, and reduce its toxic side effects. It can also improve the in vivo pharmacokinetic characteristics of the parent drug to a certain extent. Due to the poor water solubility of TP, most researchers prefer to modify the structure of TP and tend to design and synthesize new water-soluble TP derivatives with good pharmacokinetic properties in order to reduce the toxic side effects of the drug. For example, PG490-88Na, WilGraf, and Minnelide are all water-soluble TP prodrugs (NOEL PVONHOFF DD, SALUJA AK, et al. Triptolide and its derivatives as cancer therapies[J]. Trends Pharmacol Sci, 2019, 40(5):327-341). Although the aforementioned prodrugs greatly increase the water solubility of the drug by converting TP to salt, studies have found potential drawbacks such as excessively rapid degradation and release or incomplete conversion. For example, in a dose-escalation study of a Phase I clinical trial of PG490-88Na, subjects were patients with advanced solid tumors, and the administration method was once-weekly intravenous injection followed by a one-week rest period every two weeks. Common adverse reactions observed in the trial included anemia, fatigue, nausea, vomiting, diarrhea, and constipation, all rated as grade 1-2. Furthermore, pharmacokinetic studies showed that the pharmacokinetic characteristics of PG490-88Na exhibited significant inter-individual variability (2-3 times), and the degree of its conversion to TP was difficult to predict, with the conversion process being slow and incomplete.

[0005] Therefore, it is evident that PG490-88Na is not an ideal TP derivative, and the clinical trial has been forced to pause (Kitzen JJ, de Jonge MJ, Lamers CH, et al. Phase I dose-escalation study of F60008, a novel apoptsis inducer, in patients with advanced solid tumors[J]. European Journal of Cancer, 2009, 45(10): 1764-1772). Minnelide remains in Phase III clinical trials, and there have been no updates on its clinical trials since then. It is clear that whether the approach of improving water solubility by converting TP into salts can meet clinical needs remains to be discussed.

[0006] On the other hand, liposomes, as a current research and development hotspot, have shown broad application prospects. Their advantages include: reducing toxicity through targeted action while further improving efficacy; encapsulating drugs in liposomes provides significant sustained-release effects, greatly slowing the drug's elimination rate in the body, prolonging its half-life, and allowing sufficient time for the drug to target the tumor site, reducing its distribution in normal tissues, thereby improving the safety of clinical use. These characteristics are precisely the ideal effects that TP (potassium phosphate) urgently needs to achieve. Furthermore, with the development of formulation technology, many nano-formulations have been successively applied clinically, such as doxorubicin liposomes. Irinotecan liposomes (Onivyde) TM ), cytarabine liposomes mRNA liposomes, etc. (Tenchov R, Bird R, Curtze AE. Lipid Nanoparticles - From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. Published online ahead of print. ACS Nano. 2021). The above formulations fully demonstrate the feasibility of using liposomes for the market launch of anti-tumor drugs and can provide important references for the development of TP. However, due to the poor solubility, short half-life, and poor drug-likeness of TP itself, directly preparing it into liposomes presents problems such as poor drug-likeness, difficult preparation, and low encapsulation efficiency.

[0007] Therefore, combining lipid-soluble prodrugs with nano-formulations will be an important research approach to improve the drug-likeness, efficacy, and toxicity of poorly soluble drugs. For example, Mitsubishi Pharmaceutical Co., Ltd. of Japan has successfully developed and marketed dexamethasone palmitate fat emulsion injection. This product is formed by esterifying dexamethasone with palmitate to create a lipid-soluble prodrug, which is then formulated into a fat emulsion injection, increasing efficacy and reducing toxicity. Another example is Alkermes' aripiprazole lauroyl sustained-release injection, approved by the FDA in 2015. This product combines aripiprazole with fatty acids via a linking group to obtain a highly lipid-soluble aripiprazole derivative, which is then formulated into a sustained-release injection, effectively prolonging drug release and enhancing efficacy.

[0008] Chinese patent document CN105853403A discloses a paclitaxel palmitate liposome and its preparation method to address the problem of poor drug-likeness in paclitaxel nanoformulations. This patent synthesizes a series of paclitaxel fatty acid ester lipid-soluble prodrugs by esterifying paclitaxel 2'-OH with saturated fatty acids, including paclitaxel myristate, paclitaxel palmitate, and paclitaxel stearate, and then prepares them into liposomes. In vivo pharmacodynamic studies show that among the many paclitaxel lipid-soluble prodrugs, paclitaxel palmitate liposomes exhibit the strongest antitumor activity. This may be because the longer the fatty acid chain, the longer the circulation time of the paclitaxel prodrug in vivo, meaning the slower the conversion to the parent drug paclitaxel. Furthermore, prodrugs often have no or very low activity in vivo and only exert their effects after conversion to the parent drug. Therefore, liposomes with shorter chains of paclitaxel fatty acid esters (such as myristate) release and convert paclitaxel too quickly in vivo, failing to achieve sufficient long-term circulation. Conversely, liposomes with longer chains of paclitaxel fatty acid esters (such as stearate) release and convert paclitaxel too slowly in vivo, failing to reach the effective concentration of paclitaxel in vivo. Among the many paclitaxel fatty acid ester derivatives, paclitaxel palmitate achieves an appropriate balance between long-term circulation and the rate of conversion and release of paclitaxel, allowing the released active parent drug paclitaxel to maintain a high concentration in vivo for a long time. In other words, the circulation time and drug concentration of the active parent drug in vivo are maximized, thus maximizing its anti-tumor effect.

[0009] Chinese patent document CN106946975A discloses a lipid-soluble derivative of triptolide, obtained by esterification of triptolide C14-OH with eight saturated fatty acids, including butyric acid, hexanoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and stearic acid. Esterification modification significantly improves the drug-likeness of triptolide; for example, the liposome encapsulation efficiency of the eight disclosed lipid-soluble derivatives is greater than 90%, while that of triptolide liposomes is less than 50%. In vivo pharmacokinetic studies show that the biological half-lives of triptolide released in vivo by triptolide myristicate ester liposomes, triptolide palmitate ester liposomes, and triptolide stearate ester liposomes are 80.4 min, 100.9 min, and 124.2 min, respectively, significantly prolonging the half-life of triptolide. 1 / 2 =15min), laying the foundation for better anti-tumor effects in vivo.

[0010] The literature (Huang Zhiyong. Study on the effect of fatty acid chain length on the properties of triptolide prodrug liposomes [D]. 2020) developed six lipophilic derivatives of triptolide fatty acid esters, including triptolide myristate, triptolide palmitate, triptolide stearate, triptolide arachidate, triptolide behenate, and triptolide linolenic acid ester. The liposome encapsulation efficiency of these six lipophilic prodrugs was greater than 90%. In vivo antitumor efficacy studies showed that the tumor inhibition rate of the liposomes of the lipophilic prodrugs was significantly higher than that of the triptolide original drug control group. For example, the tumor inhibition rate of the triptolide behenate liposome group was 57.1%, while that of the triptolide original drug control group was 17.67%.

[0011] In summary, to improve the drug-like properties of triptolide, its lipid-soluble prodrugs and nano-formulations are important research directions, given the good stability and biocompatibility of saturated fatty acids. Therefore, triptolide saturated fatty acid esters are a key research focus. However, research on triptolide saturated fatty acid esters is still incomplete and lacks systematic and meticulous studies, particularly regarding their antitumor efficacy and safety, which require further improvement. This is especially important for triptolide, a highly active and toxic drug, where its antitumor efficacy and safety are paramount. Summary of the Invention

[0012] To address the shortcomings of existing technologies and to better develop and utilize the technology of triptolide saturated fatty acid esters, providing more effective and safer triptolide fatty acid esters and their preparations for clinical use, this invention provides triptolide lignocerate esters, their liposomes, and a preparation method thereof.

[0013] Saturated fatty acids can be classified into short-chain fatty acids (C2-C4), medium-chain fatty acids (C6-C12), long-chain fatty acids (C14-C20), and very long-chain fatty acids (C22-C26) according to the length of their carbon chains. Fu Zhiqin et al. (Chinese Patent CN106946975) disclosed saturated fatty acid esters of triptolide with a chain length of C4-C18, and Huang Zhiyong (Study on the Influence of Fatty Acid Chain Length on the Properties of Liposomes of Tripterygium Wheat Gluten Prodrug [D]. 2020) disclosed saturated fatty acid esters of triptolide with a chain length of C14-C22. To date, no research has been conducted on triptolide lignocerate (C24) and triptolide cerate (C26). However, due to its excessively long carbon chain, hexadecanoic acid (CVA) presents issues related to in vivo metabolism and safety, requiring strict control of its in vivo content. The normal blood concentration range is 0.3-0.7 μmol / L (Wei Hong. Establishment and application of GC-MS method for plasma very long chain fatty acids [J]. Clinical Pediatrics Journal, 2005(08):25-28.), which undoubtedly greatly limits its clinical application. Therefore, the inventors conducted a comparative study of triptolide ceramide ester with other related triptolide saturated fatty acid esters.

[0014] The inventors selected the preferred triptolide stearate disclosed in Chinese patent document CN106946975A, and the triptolide arachidate and triptolide behenate disclosed in the document (Huang Zhiyong. Study on the Influence of Fatty Acid Chain Length on the Properties of Liposomes of Triptolide Prodrug [D]. 2020) as controls, and synthesized a series of triptolide saturated fatty acid esters, including triptolide creosote (Example 1); and prepared them into liposome nanoformulations (Example 3); unexpectedly, it was found that triptolide creosote had the strongest antitumor effect in the in vivo antitumor efficacy study, and its tumor inhibition rate was significantly higher than that of triptolide stearate, arachidate, and behenate, and unexpectedly, its safety was also the best (Example 5); subsequent pharmacokinetic studies showed that the in vivo half-life of triptolide creosote was longer than that of other lipophilic derivatives of triptolide (Example 6). Therefore, through systematic comparative studies, this invention has found that the antitumor efficacy and safety of triptolide lignocerin ester liposomes are significantly higher than those of the previously disclosed triptolide saturated fatty acid esters. This further enhances the technical advantages of lipophilic derivatives and their nano-formulations, and further expands the drug development potential of triptolide, a highly active and highly toxic drug, demonstrating significant advantages.

[0015] In the process of synthesizing triptolide creosote, when the method of Example 1 in patent document CN106946975A was used, the synthesis yield was low (52.1%). Surprisingly, by controlling the temperature and reaction time, the solubility of creosote in the reaction system could be effectively increased, thereby obtaining a high yield of triptolide creosote (Example 7).

[0016] The primary objective of this invention is to provide a triptolide derivative, specifically a triptolide creosote ester, the chemical structure of which is shown in formula (I):

[0017]

[0018] The second objective of this invention is to provide a method for preparing the aforementioned triptolide lignocerate, which can be obtained by esterification of TP and lignocerate. The synthetic route is as follows:

[0019]

[0020] The specific preparation method is as follows: weigh out lignoceric acid and acid-binding agent and dissolve them in an appropriate amount of anhydrous DCM, and stir at room temperature to obtain a mixed solution; weigh out the catalyst and dissolve it in the above mixed solution, and stir to mix evenly; slowly add triptolide dissolved in an appropriate amount of anhydrous DCM to the above mixed solution, and continue the reaction under oil bath heating and nitrogen protection conditions; wash the reaction solution twice with saturated NaHCO3 solution and saturated NaCl solution respectively, and remove residual water with anhydrous Na2SO4 after separation, then remove DCM by rotary evaporation, and finally separate and purify by silica gel column chromatography to obtain triptolide lignoceric acid ester.

[0021] The acid-binding agent is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, p-nitrobenzoyl chloride, and N,N'-diisopropylcarbodiimide, preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0022] The catalyst is one or more of 4-pyrrolidinylpyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine and 1-hydroxybenzotriazole, preferably 4-dimethylaminopyridine.

[0023] The molar ratio of lignoceric acid to triptolide is 2:1 to 6:1, preferably 3:1 to 4:1.

[0024] The molar ratio of the acid-binding agent to triptolide is 2:1 to 6:1, preferably 3:1 to 5:1.

[0025] The oil bath temperature is 25–80°C, preferably 60–80°C.

[0026] The reaction time is 8 to 24 hours, preferably 12 to 18 hours.

[0027] The third objective of this invention is to provide a nano-formulation of triptolide creosote, wherein the nano-formulation is triptolide creosote liposomes, polymer micelles, albumin nanoparticles, fat emulsions, etc.

[0028] The nano-formulation is an injection solution or a lyophilized powder for injection, preferably a lyophilized powder for injection.

[0029] The triptolide lignocerate liposomes described herein contain triptolide lignocerate as the main drug and also include lecithin, etc.

[0030] The triptolide oxycetetracycline liposomes have a drug-lipid ratio of 1:5-1:30 (w / w) and are specifically formulated from the following recipe:

[0031]

[0032] Preferably, the drug-liposome ratio of the triptolide oxycetetracycline liposome is 1:10-1:20 (w / w), specifically formulated from the following recipe:

[0033]

[0034]

[0035] The lecithin mentioned in the above formula is selected from one or more of the following: high-purity egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, phosphatidylcholine, soybean lecithin, phosphatidylserine, myristoylphosphatidylcholine, distearylphosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; preferably, high-purity egg yolk lecithin.

[0036] The organic solvent mentioned in the above formula is selected from one or more of anhydrous ethanol, propylene glycol, tert-butanol, chloroform, and dichloromethane, with anhydrous ethanol being preferred, and the preferred amount being 0.5-5% (w / v).

[0037] The electrolyte mentioned in the above formula is selected from one or more of sodium citrate, Na2SO4, NaCl, Na2CO3, FeCl3, and Na3PO4, with NaCl being preferred, and the preferred dosage being 0-0.01% (w / v).

[0038] The freeze-drying protectant mentioned in the above formula is one or more of trehalose, sucrose, maltose, lactose, mannitol, glucose, sorbitol, xylitol, erythritol, and threonine; preferably one or more of trehalose, sucrose, and maltose, and preferably used in an amount of 10-30% (w / v).

[0039] The fourth objective of this invention is to provide a method for preparing the aforementioned triptolide oxycarboxylic acid ester liposomes.

[0040] The triptolide oxycetetracycline liposomes are prepared by injection, and are prepared by the following steps:

[0041] Weigh the prescribed amounts of triptolide oxytetracycline ester, lecithin, and DSPE-PEG2000, place them in an organic solvent for injection, and heat at 25–60°C to dissolve them, obtaining the organic phase. Weigh the prescribed amounts of electrolyte into an appropriate amount of water for injection, and heat and stir at 25–60°C to dissolve them, obtaining the aqueous phase. Under stirring conditions, slowly inject the aqueous phase into the organic phase (or slowly inject the organic phase into the aqueous phase), mix well, and obtain the crude liposomes. The crude liposomes can be emulsified by homogenizing them in a high-pressure homogenizer or by placing them in a... The liposome solution is obtained by sequentially extruding through extrusion membranes of different pore sizes in an extruder, or by high-pressure homogenization followed by extrusion. The solution is then diluted to a final volume with water for injection, filtered through a 0.22 μm filter membrane for sterilization, dispensed, and capped to obtain triptolide lignocerate liposome injection solution. Alternatively, the prescribed amount of lyophilization protectant is weighed and placed in the above liposome solution, stirred to dissolve, and diluted to a final volume with water for injection. The solution is then filtered through a 0.22 μm filter membrane for sterilization, dispensed, freeze-dried, and capped to obtain triptolide lignocerate liposome lyophilized powder for injection.

[0042] The emulsification of crude liposomes is preferably carried out by extrusion emulsification, which will result in a more uniform liposome particle size distribution. The pore size of the extruded membrane is selected from 0.8μm, 0.6μm, 0.4μm, 0.2μm, 0.1μm, and 0.05μm, and one or more extrusion methods are used to sequentially pass through the membrane from large pore size to small pore size, preferably 0.4μm, 0.2μm, 0.1μm, and 0.05μm.

[0043] The organic solvent for injection can be retained in the liposomes, or it can be removed by ultrafiltration after emulsification of the crude liposomes, or it can be removed by freeze-drying.

[0044] The electrolyte can be dissolved in an aqueous phase or in a liposome solution, preferably in an aqueous phase.

[0045] The lyophilization protectant can be dissolved in an aqueous phase or in a liposome solution.

[0046] The triptolide lignan ester liposomes have a particle size of 50-150 nm.

[0047] The aforementioned triptolide oxyphylla ester can also be prepared into nano-drug delivery formulations such as polymer micelles, albumin nanoparticles, and fat emulsions.

[0048] The fifth objective of this invention is to provide the application of the triptolide oxycarboxylate ester and the nano-formulation of the triptolide oxycarboxylate ester in the preparation of antitumor drugs.

[0049] The advantages of this invention are:

[0050] 1. Stronger efficacy: Tripterygium wilfordii is an anti-tumor drug, and anti-tumor efficacy has always been the primary factor in the screening and research of anti-tumor drugs. The tumor inhibition rate of Tripterygium wilfordii liposome is not only higher than that of other saturated fatty acid esters of Tripterygium wilfordii (Example 5), but also higher than that of Minnelide (Example 55) in the current phase III clinical trials abroad, showing a significant efficacy advantage.

[0051] 2. Lower toxicity: The high toxicity of triptolide severely limits its clinical application, while in vitro and in vivo studies of triptolide lignocellulosic acid ester liposomes have shown that its toxicity is significantly lower than that of other lipid-soluble prodrugs of triptolide, demonstrating a clear safety advantage.

[0052] 3. Stronger sustained-release effect and longer half-life: Tripterygium wilfordii has a short half-life (15 min) and is rapidly eliminated in vivo. In in vitro release studies, the sustained-release effect of triptolide lignocellulosic acid ester liposomes is significantly stronger than that of other lipid-soluble prodrugs of triptolide (Example 4). In in vivo pharmacokinetic studies, the in vivo half-life of triptolide lignocellulosic acid ester liposomes is longer than that of other lipid-soluble prodrugs of triptolide (Example 6).

[0053] 4. Smoother formulation process: In the formulation research of this invention, the liposomes are made smaller and more uniform in size by controlling the amount of organic solvent (Example 16), optimizing the ratio of drug to lipid (Example 17), and adding an appropriate amount of electrolyte (Example 18), resulting in a smoother preparation process. Attached Figure Description

[0054] Figure 1 This is an in vitro release diagram of different triptolide derivative liposomes in Example 4.

[0055] Figure 2 This is a graph showing the change in rat blood drug concentration over time in Example 6.

[0056] Figure 3 This is a visual representation of the isolated tumors of mice in each group in Example 55.

[0057] Figure 4 The images show the tail vein irritation of mice in each group after drug administration in Example 55.

[0058] Figure 5 This is a diagram of vascular irritation in Example 56. Detailed Implementation

[0059] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0060] Example 1: Preparation of Tripterygium wilfordii derivatives

[0061] 1. Experimental prescription

[0062] Table 1. Recipe Design for Experimental Validation Scheme

[0063] TP 1.0 mmol TP TP TP TP Saturated fatty acids 3.0 mmol stearic acid Arachidonic acid behenic acid Ciliate N,N'-Dicyclohexylcarbodiimide 3.0 mmol DCC DCC DCC DCC 4-Dimethylaminopyridine 3.0 mmol DMAP DMAP DMAP DMAP Anhydrous DCM 20mL DCM DCM DCM DCM

[0064] 2. Preparation process

[0065] According to the preparation method of Example 1 of patent CN106946975, the prescribed amounts of saturated fatty acid, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were added to the reaction vessel, and 20 mL of anhydrous DCM was added and stirred to dissolve. The mixture was stirred for 30 min under ice bath conditions. The prescribed amount of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added dropwise to the reaction system. The reaction was carried out for 30 min under ice bath conditions and continued to react overnight at room temperature. The reactants were then separated and purified by silica gel column chromatography.

[0066] 3. Experimental Results

[0067] (1) Tripterygium wilfordii stearate TP-SA was obtained as a light yellow solid powder, 519.3 mg. Yield: 82.90%.

[0068] ESI-MS (m / z): 627.43 [M+H] + ;

[0069] 1 H NMR (400MHz, CDCl3) δ5.09 (s, 1H), 4.68 (s, 2H), 3.83 (d, J = 2.9Hz, 1H), 3.53 (d, J = 2 .6Hz,1H),3.46(d,J=5.5Hz,1H),2.70(d,J=12.6Hz,1H),2.54–2.25(m,3H),2.24–2 .08(m,2H),1.90(dd,J=16.5,10.3Hz,3H),1.75–1.62(m,2H),1.57(dd,J=12.3,4.8 Hz,1H),1.45–1.11(m,28H),1.06(s,3H),0.96(d,J=6.9Hz,3H),0.92–0.78(m,6H).

[0070] 13C NMR (101MHz, CDCl3) δ173.41(2C),160.28,125.70,70.79,70.17,63.75,63.52,61.23,59.89,55.49,55.15,40.54,35.85,34. 52,32.09,29.98,29.92–29.75(8C),29.65,29.50(2C),29.17,28.27,25.18,23.62,22.86,17.52,17.05,16.71,14.12,13.68.

[0071] The chemical structure is shown in formula (II) below:

[0072]

[0073] (2) Tripterygium wilfordii arachidate TP-EA was obtained as a light yellow solid powder, 532.8 mg. Yield: 81.79%.

[0074] ESI-MS (m / z): 652.44 [M+H] + ;

[0075] 1 H NMR (400MHz, CDCl3) δ5.02(s,1H),4.71–4.49(m,2H),3.76(d,J=3.0Hz,1H),3.46(d,J=2.8 Hz,1H),3.39(d,J=5.6Hz,1H),2.70–2.55(m,1H),2.50–2.20(m,3H),2.20–1.98(m,2H),1. 94–1.72(m,2H),1.70–1.56(m,3H),1.52(d,J=4.9Hz,1H),1.49(d,J=4.9Hz,1H),1.36–1.1 0(m,31H),0.99(s,3H),0.89(d,J=7.0Hz,3H),0.81(t,J=6.8Hz,3H),0.77(d,J=6.9Hz,3H).

[0076] 13C NMR(101MHz, CDCl3)δ173.47(2C),160.26,125.80,70.82,70.20,63.80,63.59,61.27,59.94,55.55,55.20,40.60,35.90,34.58 ,32.14,30.04,29.99–29.81(11C),29.71(s),29.56(2C),29.23,28.31,25.23,23.69,22.91,17.75,17.28,16.94,14.35,13.90.

[0077] The chemical structure is shown in formula (III) below:

[0078]

[0079] (3) Tripterygium wilfordii behenate TP-BA was obtained as a white solid powder, 594.7 mg. Yield: 87.15%.

[0080] ESI-MS (m / z): 683.49 [M+H] + ;

[0081] 1 H NMR (400MHz, CDCl3) δ5.02(s,1H),4.83–4.39(s,2H),3.75(d,J=3.1Hz,1H),3.46(d,J= 2.7Hz,1H),3.39(d,J=5.6Hz,1H),2.70–2.55(m,1H),2.33(tdd,J=23.6,17.7,11.8Hz,3 H),2.17–1.99(m,2H),1.91–1.75(m,2H),1.68–1.55(m,2H),1.50(dd,J=12.4,4.8Hz,1H ),1.37–1.08(m,36H),0.99(s,3H),0.89(d,J=7.0Hz,3H),0.78(dd,J=12.4,7.0Hz,6H).

[0082] 13C NMR(101MHz, CDCl3)δ173.41(2C),160.28,125.69,70.79,70.16,63.75,63.51,61.23,59.89,55.49,55.15,40.54,35.84,34.5 2,32.09,29.98,29.93–29.75(12C),29.65,29.50(2C),29.17,28.27,25.18,23.62,22.86,17.69,17.23,16.88,14.29,13.85.

[0083] The chemical structure is shown in formula (IV) below:

[0084]

[0085] (4) Tripterygium wilfordii ester was obtained as a light yellow solid powder, 370.7 mg. Yield: 52.12%.

[0086] ESI-MS (m / z): 711.51 [M+H] + ;

[0087] 1 H NMR (400MHz, CDCl3) δ7.20 (s, 2H), 5.02 (s, 2H), 4.60 (s, 4H), 3.75 (d, J = 3.0Hz, 2H), 3.46 (d, J = 2.4Hz, 2H), 3.39(d,J=5.6Hz,2H),2.89(s,1H),1.19(s,101H),0.99(s,7H),0.89(d,J=7.0Hz,7H),0.84–0.71(m,15H).

[0088] 13C NMR (101MHz, CDCl3) δ173.21 (d, J = 4.4Hz), 159.97 (s), 125.63 (s), 81.55–81.35 (m), 77.28 (d, J = 11. 5Hz),77.02(s),76.70(s),70.64(s),69.94(s),63.60(s),63.39(s),61.04(s),59.74(s),55.33(s) ),55.01(s),40.41(s),35.71(s),34.37(s),31.92(s),30.00–29.17(m),29.06(d,J=8.7Hz),28.15 (s),25.02(s),24.79(s),23.50(s),22.69(s),17.52(s),17.08(s),16.74(s),14.10(s),13.69(s).

[0089] The chemical structure is shown in the following formula (V):

[0090]

[0091] 4. Results Analysis

[0092] Mass spectrometry, proton NMR, and carbon NMR spectroscopy confirmed that, following the preparation method in Example 1 of patent CN106946975, saturated fatty acids can be bonded to the C14-OH of TP using an acid-binding agent and a catalyst, thus successfully synthesizing a series of triptolide derivatives. However, the patented method showed that the synthesis yield of triptolide lignocerate was low (only 52.1%).

[0093] Example 2: Determination of logP values ​​of triptolide and its derivatives

[0094] 1. Sample Source

[0095] TP-SA, TP-EA, TP-BA and TP-LA prepared from formulations 1, 2, 3 and 4 in Example 1 were used as triptolide derivatives, with TP as a control.

[0096] 2. Determination of logP value

[0097] The log P value was determined by the shake-flask method. First, specific amounts of water and n-octanol were added to separate funnels, shaken well, and separated to obtain a water-saturated n-octanol solution and a water-saturated n-octanol aqueous solution. Quantitatively and accurately weighed triptolide and its derivatives into volumetric flasks containing water-saturated n-octanol solution, dissolved by sonication, and diluted dropwise with water-saturated n-octanol solution to volume. 0.5 mL of this solution was transferred to a 5 mL volumetric flask, diluted with MeOH solution, and diluted to volume. The concentration of triptolide and its derivatives was determined according to the content determination method. The above solution was accurately measured into a vial, and 10 times the volume of water-saturated n-octanol aqueous solution was added. The solution was shaken for one day at 37°C and 75 rpm in a constant temperature water bath to allow triptolide and its derivatives to reach equilibrium in the aqueous and organic phases. Transfer the n-octanol phase to a centrifuge tube and centrifuge at 3000 rpm for 10 min. Accurately measure the n-octanol phase into a volumetric flask, dilute it 10 times with MeOH solution, and determine the content of triptolide and its derivatives in the n-octanol phase according to the content determination method. Drug concentration in aqueous phase = total drug concentration - drug concentration in n-octanol phase.

[0098]

[0099] 3. In this embodiment, the content of triptolide and its derivatives was determined by high performance liquid chromatography (2020 edition of Chinese Pharmacopoeia, Part IV, General Chapter 0512), as follows:

[0100] Chromatographic conditions: Agilent ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm), mobile phase: 100% MeOH, column temperature: 30 ℃, flow rate: 1.0 mL / min, injection volume: 20 μL, detection wavelength: 218 nm.

[0101] Preparation of reference solution: Accurately weigh 10 mg of triptolide and its derivatives, place them in a 10 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use as the reference stock solution.

[0102] Preparation of the standard curve: Accurately pipette different volumes of the reference standard stock solution and dilute them to obtain a series of solutions with mass concentrations of 5.00, 10.00, 25.00, 50.00, 100.00, 250.00, and 500.00 μg / mL. Inject 20 μL of each solution into the high-performance liquid chromatography system and analyze according to the "Chromatographic Conditions". Perform linear regression with concentration C (μg / mL) on the x-axis and peak area A on the y-axis to obtain the standard curve.

[0103] Assay: Accurately pipette 1 mL of each triptolide and its derivative sample solution into a 5 mL volumetric flask, dilute to the mark with methanol, and vortex to mix thoroughly. Accurately inject 20 μL of each solution into the high-performance liquid chromatograph, record the chromatogram, and calculate the result by peak area according to the standard curve method.

[0104] 4. Experimental Results

[0105] Table 2. Results of log P value determination for TP and different TP derivatives

[0106] Tripterygium wilfordii 0.52±0.01 1.92 Tripterygium wilfordii stearate 2.41±0.04 1.66 Tripterygium wilfordii arachidonic acid ester 2.57±0.02 0.78 Tripterygium oxyphylla behenate 2.78±0.04 1.44 Tripterygium wilfordii ester 3.12±0.03 0.96

[0107] 5. Results Analysis

[0108] Compared with TP-SA, TP-EA and TP-BA, the TP-LA prepared in Example 1 has a higher log P value, and the TP-LA prepared by modifying TP with lignosulfonic acid has the strongest lipophilicity.

[0109] Example 3: Liposomes prepared from different triptolide derivatives

[0110] 1. Preparation of liposomes of different triptolide derivatives

[0111] Table 3. Recipe Design for Experimental Validation Scheme

[0112]

[0113] 2. Preparation process

[0114] Following the preparation method in Example 2 of patent CN106946975A, 0.2g of triptolide derivative, 2g of egg yolk phospholipid, 0.2g of PEGylated distearate ethanolamine, and 0.2g of cholesterol were weighed and dissolved in an appropriate amount of DCM. The solvent was removed by vacuum rotary evaporation at 45°C to obtain a lipid film. The film was then hydrated with 80mL of water for injection to obtain crude liposomes. The crude liposomes were homogenized and emulsified in a high-pressure homogenizer to obtain a liposome solution. 25g of trehalose was weighed and dissolved in the above liposome solution. The solution was diluted to 100mL with water for injection, and the pH was adjusted to 6.0 with sodium hydroxide and hydrochloric acid. The solution was filtered through a 0.22μm filter membrane, dispensed, freeze-dried, and capped to obtain the final product.

[0115] 3. Experimental Results

[0116] The appearance of the liposomes prepared from the above-mentioned different injectable triptolide derivatives after reconstitution and their filtration smoothness through a 0.22 μm filter membrane were investigated. The results are shown in Table 4 below.

[0117] Table 4. Preparation results of liposomes with different triptolide derivatives

[0118]

[0119] 4. Results Analysis

[0120] Experimental results show that liposomes have a good encapsulation effect on lipid-soluble TP derivatives, with a particle size distribution of approximately 120 nm.

[0121] Example 4: In vitro release of different triptolide derivatives via liposomes

[0122] 1. Sample Source

[0123] TP-SA liposomes, TP-EA liposomes, TP-BA liposomes and TP-LA liposomes prepared from formulations 1, 2, 3 and 4 in Example 3 were used as liposome formulations.

[0124] 2. Design of in vitro release protocols for liposomes containing different triptolide derivatives

[0125] The release kinetics of triptolide derivative liposomes were quantitatively determined through a drug release assay. Unlike previous methods that used aqueous media, this experiment employed an oily substance, n-octanol, as the release medium. 100 mL of the release medium was added to a 200 mL dissolution vessel. The mixture was heated and stirred thoroughly at 37 ± 0.5 °C and a stirring speed of 100 rpm before drug administration. A 1 mL sample of liposomes was then pipetteed into a dissolution vessel containing an appropriate volume of release medium. The mixture was stirred at a constant temperature and speed. 2 mL samples were taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and fresh release medium was immediately added. The samples were then filtered through a 0.22 μm nylon membrane. 100 μL of the test solution was accurately pipetted into a 5 mL volumetric flask, diluted with methanol, and brought to volume. After vortexing for 1 min, a 20 μL sample was injected for content determination, and the in vitro cumulative release rate (Er) was calculated.

[0126]

[0127] Among them, C n : Sample concentration at each time point; L: Formulation labeled amount; V1: Fixed sampling volume at each time point; V2: Release medium volume.

[0128] 3. Results of cumulative drug release in vitro

[0129] Table 5. In vitro release results of liposomes containing different triptolide derivatives

[0130] 0.5h 29.41±1.45 21.45±1.99 17.89±1.23 11.02±0.99 1h 76.80±7.56 68.61±8.04 27.80±8.48 19.85±3.29 2h 90.52±6.02 82.09±2.45 45.45±6.19 32.14±2.06 4h 95.70±5.47 92.58±3.12 66.09±4.20 54.28±5.04 6h 95.69±6.34 95.87±4.55 80.77±7.21 67.80±2.45 8h 97.87±6.55 95.88±5.31 92.06±9.46 90.82±6.45 12h 98.00±7.09 96.65±4.80 96.80±2.89 96.88±8.36 24h 99.04±4.28 98.80±6.67 97.01±5.08 97.41±7.57

[0131] 4. Results Analysis

[0132] TP-SA, TP-EA, TP-BA, and TP-LA liposomes prepared using the method in Example 3 were selected for in vitro release comparison. The experimental results showed that all the prepared triptolide derivative liposomes could release the drug completely in n-octanol. However, TP-SA and TP-EA liposomes showed a cumulative release rate of over 80% after 2 hours, exhibiting a significant burst release phenomenon. Compared to other TP derivative liposomes, TP-LA liposomes showed a more pronounced sustained-release effect.

[0133] Example 5: In vivo antitumor effect of triptolide derivative liposomes

[0134] Based on the triptolide derivative liposomes prepared in Example 3, an antitumor study was conducted on the Panc 02 pancreatic cancer model, using TP (DMSO) solution and TP-SA liposomes as controls. The experimental design and results are as follows:

[0135] 1. Sample Source

[0136] Take TP (DMSO) solution as a positive control drug; take TP-SA liposomes, TP-EA liposomes, TP-BA liposomes and TP-LA liposomes prepared by formulations 1, 2, 3 and 4 in Example 3 as liposome preparations.

[0137] 2. Establishment of a mouse Panc 02 pancreatic cancer tumor model and design of a drug administration regimen

[0138] Mouse pancreatic cancer Panc 02 cells were cultured in DMEM medium at 37°C under standard conditions of 5% CO2, and passaged on average every two days. At the logarithmic growth phase, the cell concentration was adjusted to 1×10⁻⁶ cells using culture medium. 7 Panc02 cells were subcutaneously inoculated into the right axilla of ICR mice under aseptic conditions at a rate of 100-300 mm² / mL to establish a mouse pancreatic cancer model. 3 Mice were randomly divided into 6 groups (n=6 per group) based on tumor volume. These groups included a control group, a TP (DMSO) solution group, and TP-SA, TP-EA, TP-BA, and TP-LA liposome dosage groups. The drugs were administered via tail vein injection at a dose of 0.6 mg / kg (based on TP). The control group received 0.2 mL of physiological saline. Drug administration was repeated every two days for a total of four doses. Mice were sacrificed on the third day after drug withdrawal, and their body weight, tumor removal, and weighing were performed to calculate the tumor inhibition rate.

[0139]

[0140] 3. Anti-tumor effects

[0141] Using the mouse Panc 02 pancreatic cancer model, the antitumor effects of TP derivative liposomes and TP (DMSO) solution were investigated. The results are shown in Table 6.

[0142] Table 6. Comparison of the antitumor effects of TP and TP derivative liposomes

[0143] Model group / 16.70 0.878±0.231 / TP solution group 0.60 mg / kg -5.62 0.517±0.097 41.12％ TP-SA liposomes 1.04 mg / kg 9.39 0.452±0.129 48.52％ TP-EA liposomes 1.08 mg / kg 10.34 0.390 ± 0.093 * ]] 55.58％ TP-BA liposomes 1.14 mg / kg 15.87 0.288 ± 0.090 *# ]] 67.20％ TP-LA liposomes 1.18 mg / kg 16.95 <![CDATA[0.168±0.063 **## ]]> 80.86％

[0144] Note: Compared to the model group * P < 0.05 ** P < 0.01; compared with the TP group, # P < 0.05 ## P < 0.01.

[0145] 4. Results Analysis

[0146] TP-SA liposomes, TP-EA liposomes, TP-BA liposomes, and TP-LA liposomes prepared according to the method in Example 3 were used for pharmacodynamic evaluation in a mouse Panc 02 pancreatic cancer model, along with TP solution. The results showed that the efficacy of the prepared TP-LA liposomes was significantly superior to that of TP solution and other TP derivative liposomes. Mice administered with TP solution experienced a significant decrease in body weight, and intravenous injection of TP solution, TP-SA liposomes, TP-EA liposomes, and TP-BA liposomes resulted in significant tail swelling and ulceration, indicating significant irritation. Mice treated with TP-LA liposomes showed no significant abnormalities and exhibited a normal weight gain trend, demonstrating that the liposomes prepared in this invention significantly improve both overall efficacy and biosafety.

[0147] Example 6: Pharmacokinetic Study in Rats

[0148] 1. Sample Source

[0149] Take TP (DMSO) solution as a positive control; take TP derivative liposomes prepared in Example 3 as liposome samples.

[0150] 2. Rat Pharmacokinetic Experiment Design

[0151] The rats were administered the drug via the tail vein at a dose of equimolar TP 0.6 mg / kg. Blood was collected at 6 min, 12 min, 18 min, 30 min, 48 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 5 h, 12 h, 24 h, and 72 h. Five SD rats (male) were used in each group.

[0152] Sample processing and analysis methods: Protein was precipitated in plasma by adding 4 times the volume of methanol and centrifuged at 12000 rpm for 10 min. The supernatant was collected, and the methanol was evaporated. The remaining liquid was extracted twice with 400 μL of methyl tert-butyl ether and centrifuged at 12000 rpm for 10 min. The organic extract phase was collected, concentrated to dryness, and reconstituted with 100 μL of methanol. The supernatant was centrifuged and injected into an LC-MS / MS system to determine the content and calculate the blood drug concentration. Pharmacokinetic fitting was performed using DAS 2.0 software. The pharmacokinetic parameters are shown in Table 7.

[0153] 3. Pharmacokinetic parameters

[0154] Table 7. Pharmacokinetic results of TP solution and TP derivative liposomes metabolizing TP.

[0155] <![CDATA[C max ]]> ng / L 697.686±67.12 441.125±35.18 376.125±46.19 342.378±76.28 293.1223±40.87 <![CDATA[AUC 0-∞ ]]> ng / L*h 336.569±37.60 704.315±68.94 976.253±67.03 1488.436±120.08 2719.967±168.42 <![CDATA[t 1 / 2 ]]> h 0.220±0.04 1.349±0.13 3.315±0.23 8.166±0.45 9.651±1.74 V L / kg 566.793±70.24 1658.397±101.32 2940.011±110.08 4750.123±146.23 3071.977±230.610 CL L / kg / h 1782.698 851.892 614.595 403.108 220.591 MRT h 0.547±0.06 3.583±0.48 6.246±0.89 25.421±1.21 23.790±2.130

[0156] 4. Results Analysis

[0157] Experimental results show that, compared with other TP derivative liposomes and TP solutions, TP-LA liposomes metabolize the parent drug TP with a larger AUC and a longer half-life.

[0158] In conclusion, the triptolide lignocerate ester prepared by modifying TP with lignocerate has a longer in vivo duration of action and a more significant effect on enhancing in vivo antitumor efficacy, as well as lower administration irritation and toxicity compared to other triptolide derivatives.

[0159] Example 7: The critical role of reaction temperature in the development of triptolide oxycephalin prodrug

[0160] 1. Experimental prescription

[0161] Table 8. Recipe Design for Experimental Validation Scheme

[0162]

[0163] 2. Preparation process

[0164] In a reaction vessel, add the prescribed amount of lignoceric acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, add an appropriate amount of anhydrous DCM and stir to dissolve. Stir for 30 min at room temperature, then add the prescribed amount of 4-dimethylaminopyridine to the solution and mix well. Dissolve the prescribed amount of TP in an appropriate amount of anhydrous DCM and slowly add it to the reaction system. Heat in an ice bath or oil bath and continue the reaction for 12 h under nitrogen protection. Wash the reaction solution twice with saturated NaHCO3 solution and saturated NaCl solution before and after, respectively. After separation, remove residual water with anhydrous Na2SO4, then remove DCM by rotary evaporation, and finally purify by silica gel column chromatography.

[0165] 3. Experimental Results

[0166] The appearance and yield of the above-mentioned triptolide oxyphylla ester prodrug were investigated, and the results are shown in Table 9 below:

[0167] Table 9. Effect of reaction temperature on the preparation of triptolide wood wax ester.

[0168] Prescription 1 Light yellow solid powder 0.350 49.3 Prescription 2 off-white solid powder 0.633 89.1 Prescription 3 off-white solid powder 0.657 92.4 Prescription 4 off-white solid powder 0.677 95.3 Prescription 5 off-white solid powder 0.679 95.6

[0169] 4. Results Analysis

[0170] Experimental results show that controlling different reaction temperatures has a significant impact on the yield of triptolide lignocerate derivatives. At lower reaction temperatures, lignoceric acid cannot be completely dissolved in the reaction system, leading to a decrease in the synthesis yield. Conversely, when the reaction temperature increases, lignoceric acid dissolves well, and the synthesis yield of triptolide lignocerate is significantly improved. However, considering that excessively high temperatures may introduce reaction impurities and result in high energy loss, controlling the appropriate reaction temperature is crucial to this invention.

[0171] Example 8: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0172] In a reaction vessel, 4.0 mmol of lignoceric acid and 5.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of 4-dimethylaminopyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 24 h in an oil bath at 70 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.685 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 96.4%.

[0173] Example 9: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0174] 2.0 mmol of lignoceric acid and 3.0 mmol of N,N'-dicyclohexylcarbodiimide were added to a reaction vessel, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 3.0 mmol of 4-dimethylaminopyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 12 h in an oil bath at 40 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.662 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 93.2%.

[0175] Example 10: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0176] In a reaction vessel, 6.0 mmol of lignoceric acid and 2.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 3.0 mmol of 4-pyrrolidinylpyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 8 h in an oil bath at 25 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.654 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 92.1%.

[0177] Example 11: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0178] In a reaction vessel, 4.0 mmol of lignoceric acid and 6.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of 4-dimethylaminopyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 10 h in an oil bath at 30 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.666 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 93.8%.

[0179] Example 12: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0180] In a reaction vessel, 3.0 mmol of lignoceric acid and 4.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of 4-pyrrolidinylpyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 16 h in a 60°C oil bath under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.679 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 95.5%.

[0181] Example 13: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0182] In a reaction vessel, 3.0 mmol of lignoceric acid and 4.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of N,N-diisopropylethylamine and 2.0 mmol of 1-hydroxybenzotriazole were added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 8 h in an oil bath at 80 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.655 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 92.2%.

[0183] Example 14: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0184] In a reaction vessel, 3.0 mmol of lignoceric acid and 2.0 mmol of N,N'-dicyclohexylcarbodiimide were added, along with an appropriate amount of anhydrous DCM, and stirred until dissolved. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of 4-dimethylaminopyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 16 h in a 40°C oil bath under nitrogen protection. The reaction solution was washed twice, first with saturated NaHCO3 solution and then with saturated NaCl solution. After separation, residual water was removed with anhydrous Na2SO4, followed by rotary evaporation to remove DCM. Finally, 0.661 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 93.0%.

[0185] Example 15: Preparation of Tripterygium wilfordii lacrylate (TP-LA)

[0186] 5.0 mmol of lignoceric acid and 5.0 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to a reaction vessel, and an appropriate amount of anhydrous DCM was added and stirred to dissolve. The mixture was stirred at room temperature for 30 min, and then 4.0 mmol of 4-pyrrolidinylpyridine was added to the solution and mixed thoroughly. 1.0 mmol of TP was dissolved in an appropriate amount of anhydrous DCM and slowly added to the reaction system. The reaction was continued for 18 h in an oil bath at 70 °C under nitrogen protection. The reaction solution was washed twice with saturated NaHCO3 solution and saturated NaCl solution, respectively. After separation, residual water was removed with anhydrous Na2SO4, and DCM was removed by rotary evaporation. Finally, 0.686 g of triptolide lignoceric acid ester was obtained by silica gel column chromatography. The yield was 96.6%.

[0187] Example 16: The criticality of the amount of organic solvent used for injection in the development of triptolide lignocerate liposomes

[0188] 1. Experimental prescription

[0189] Table 10. Recipe Design for Experimental Validation Scheme

[0190] Tripterygium wilfordii ester 0.30g 0.30g 0.30g 0.30g 0.30g High-purity egg yolk lecithin 3.00g 3.00g 3.00g 3.00g 3.00g DSPE-PEG2000 0.10g 0.10g 0.10g 0.10g 0.10g Anhydrous ethanol 0.50g 3.00g 5.00g 10.00g 15.00g Water for Injection Up to 100mL Up to 100mL Up to 100mL Up to 100mL Up to 100mL

[0191] 2. Preparation process

[0192] Weigh the prescribed amounts of triptolide lignocerate, high-purity egg yolk lecithin, and DSPE-PEG2000, and dissolve them in the prescribed amount of anhydrous ethanol at 60°C to obtain the organic phase. Weigh an appropriate amount of water for injection and heat it at 60°C to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Weigh 25g of sucrose and place it in the above liposome solutions, stir to dissolve, and dilute to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, lyophilize, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0193] 3. Experimental Results

[0194] The appearance of the liposomes and the filtration smoothness of the 0.22 μm filter membrane were investigated, and the results are shown in Table 11 below:

[0195] Table 11 Results of the effect of the amount of organic solvent used for injection on TP-LA liposomes

[0196]

[0197] The appearance and reconstitution properties of the lyophilized powder injections after direct freeze-drying of the above liposome solutions were examined, and the results are shown in Table 12 below:

[0198] Table 12 Results of the effect of the amount of organic solvent used for injection on the freeze-drying of TP-LA liposomes

[0199] Prescription 1 Shrinkage, collapse 128 147.5 0.193 Prescription 2 flat and full 34 102.2 0.141 Prescription 3 Shrinkage, peeling 52 112.6 0.152 Prescription 4 spray bottle / / / Prescription 5 spray bottle / / /

[0200] 4. Results Analysis

[0201] Experimental results show that different amounts of injectable organic solvents have a significant impact on the preparation and particle size distribution of triptolide lignocellulosic acid ester liposomes. When the content of injectable organic solvent is low, the lipids cannot be completely dissolved even under heating, affecting the drug-like properties of the liposomes and increasing the extrusion pressure. Conversely, when the content of injectable organic solvent is high, the particle size of the prepared triptolide lignocellulosic acid ester liposomes increases, and the liposome solution can be directly freeze-dried into lyophilized powder for injection vials. Therefore, the appropriate amount of injectable organic solvent is crucial to this invention.

[0202] Example 17: Comparative Evaluation of Tripterygium wilfordii liposomes prepared with different drug-liposome ratios

[0203] 1. Preparation of Tripterygium wilfordii liposomes with different drug-lipid ratios

[0204] Table 13. Regimen Design for Validation Protocol

[0205] Tripterygium wilfordii ester 0.30g 0.30g 0.30g 0.30g 0.30g High-purity egg yolk lecithin 1.50g 3.00g 4.50g 6.00g 9.00g DSPE-PEG2000 0.10g 0.10g 0.10g 0.10g 0.10g Water for Injection Up to 100mL Up to 100mL Up to 100mL Up to 100mL Up to 100mL Remark Drug-to-lipid ratio 1:5 Drug-to-lipid ratio 1:10 Drug-to-lipid ratio 1:15 Drug-to-lipid ratio 1:20 Drug-to-lipid ratio 1:30

[0206] 2. Preparation process

[0207] Weigh the prescribed amounts of triptolide lignocerate, high-purity egg yolk lecithin, and DSPE-PEG2000, and dissolve them in 3g of anhydrous ethanol by heating at 60℃ to obtain the organic phase. Weigh an appropriate amount of water for injection and heat it at 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain the crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Weigh 25g of sucrose and place it in the above liposome solutions, stir to dissolve, and dilute to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0208] 3. Experimental Results

[0209] The extrusion pressure, liposome appearance, and particle size distribution during the preparation of the above liposomes were investigated, and the results are shown in Table 14 below.

[0210] Table 14 Results of the effect of different drug-liposome ratios on triptolide lignan liposomes

[0211] Prescription 1 A homogeneous solution with a pale blue opalescence and semi-transparent appearance. 149.10 0.280 150 bar Prescription 2 A homogeneous solution with a pale blue opalescence and semi-transparent appearance. 105.81 0.143 10 bar Prescription 3 A homogeneous solution with a pale blue opalescence and semi-transparent appearance. 110.28 0.178 30 bar Prescription 4 A homogeneous solution with a pale blue opalescence and semi-transparent appearance. 114.82 0.228 80 bar Prescription 5 A homogeneous solution with a pale blue opalescence and semi-transparent appearance. 125.47 0.241 130 bar

[0212] 4. Results Analysis

[0213] Experimental results showed that different drug-to-lipid ratios had a significant impact on the particle size distribution and extrusion pressure of triptolide lignocellulosic acid liposomes during preparation. When the drug-to-lipid ratio was high, the extrusion pressure, particle size, and PDI of the liposomes increased significantly. However, increasing the phospholipid ratio and decreasing it to between 1:10 and 1:20 did not result in a significant change in the particle size distribution of the prepared triptolide lignocellulosic acid liposomes. Therefore, we optimally selected a drug-to-lipid ratio of 1:10–1:20.

[0214] Example 18: Comparative Evaluation of Tripterygium wilfordii liposomes prepared with different electrolyte dosages

[0215] 1. Preparation of triptolide lignocellulose liposomes with different electrolyte dosages

[0216] Table 15. Regimen Design for Validation Protocol

[0217] Tripterygium wilfordii methyl ester 0.30g 0.30g 0.30g 0.30g 0.30g High-purity egg yolk lecithin 3.00g 3.00g 3.00g 3.00g 3.00g DSPE-PEG2000 0.10g 0.10g 0.10g 0.10g 0.10g NaCl 0 10μg 50μg 100μg 500μg Water for Injection Up to 100mL Up to 100mL Up to 100mL Up to 100mL Up to 100mL

[0218] 2. Preparation process

[0219] Weigh the prescribed amounts of triptolide lignocerate, high-purity egg yolk lecithin, and DSPE-PEG2000, place them in 3g of anhydrous ethanol, and heat at 60℃ to dissolve, obtaining the organic phase; weigh the prescribed amount of NaCl, place it in an appropriate amount of water for injection, and heat and stir at 60℃ to dissolve, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm respectively to obtain liposome solutions; weigh 25g of sucrose and place it in the above liposome solutions, stir to dissolve, and dilute to 100mL with water for injection, filter through a 0.22μm filter membrane for sterilization, dispense, freeze-dry, and cap, to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0220] 3. Experimental Results

[0221] The extrusion pressure, liposome appearance, and filtration smoothness of the 0.22 μm filter membrane during the preparation of the above liposomes were investigated, and the results are shown in Table 16 below.

[0222] Table 16 Results of the effect of different electrolyte dosages on triptolide lignan liposomes

[0223]

[0224] 4. Results Analysis

[0225] Experimental results showed that different electrolyte dosages had a significant impact on the particle size distribution of triptolide lignocellulosic acid liposomes. When electrolytes were added to the liposomes, the particle size and PDI (particulate density index) decreased significantly. However, when the electrolyte content was high, the ionic strength of the prepared triptolide lignocellulosic acid liposomes tended to saturate, and the particle size distribution showed no significant change. Therefore, adding an appropriate amount of electrolyte to the liposomes is one of the technical features of this invention.

[0226] Example 19: Preparation of Tripterygium wilfordii liposomes

[0227] Weigh 0.5g of triptolide lignocerate, 10g of high-purity egg yolk lecithin, and 1g of DSPE-PEG2000 according to the prescription amount, and place them in 10g of anhydrous ethanol. Heat at 25℃ to dissolve, obtaining the organic phase. Weigh 50μg of NaCl, place it in an appropriate amount of water for injection, and heat and stir at 25℃ to dissolve, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Homogenize the crude liposomes twice each at 15000psi, 20000psi, and 25000psi using a high-pressure homogenizer to obtain liposome solutions. Add an equal amount of water for injection and ultrafilter five times to remove the organic solvent. Make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0228] The average particle size was measured to be 116.1 nm, and the PDI was 0.101.

[0229] Example 20: Preparation of Tripterygium wilfordii liposomes

[0230] Weigh 0.05g of triptolide lignocerate and 1g of hydrogenated soybean lecithin according to the prescription and place them in 0.1g of anhydrous ethanol. Heat at 60℃ to dissolve them to obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain the crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Make up to 100mL with water for injection, filter through a 0.22μm filter membrane for sterilization, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0231] The average particle size was measured to be 75.8 nm, and the PDI was 0.116.

[0232] Example 21: Preparation of Tripterygium wilfordii liposomes

[0233] Weigh 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.4g of DSPE-PEG2000 according to the prescription and place them in 5g of anhydrous ethanol. Heat at 60℃ to dissolve and obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Add an equal amount of water for injection and ultrafilter four times to remove the organic solvent. Make up the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0234] The average particle size was measured to be 108.9 nm, and the PDI was 0.146.

[0235] Example 22: Preparation of Tripterygium wilfordii liposomes

[0236] Weigh out 3g of triptolide lignocerate, 9g of hydrogenated soybean lecithin, 6g of dipalmitoylphosphatidylcholine, and 0.1g of DSPE-PEG2000 according to the prescription, and place them in 15g of anhydrous ethanol. Heat at 35℃ to dissolve them to obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 35℃ to obtain the aqueous phase. Slowly inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm respectively to obtain liposome solutions. Add an equal amount of water for injection and ultrafilter 7 times to remove the organic solvent. Make up the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0237] The average particle size was measured to be 148.7 nm, and the PDI was 0.188.

[0238] Example 23: Preparation of Tripterygium wilfordii liposomes

[0239] Weigh out 0.1g of triptolide lignocerate, 1g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription, and place them in 2g of anhydrous ethanol. Heat at 55℃ to dissolve, obtaining the organic phase. Weigh out an appropriate amount of water for injection and heat at 55℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Homogenize the crude liposomes twice each at 15000psi, 20000psi, and 25000psi using a high-pressure homogenizer to obtain liposome solutions. Make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0240] The average particle size was measured to be 70.1 nm, and the PDI was 0.141.

[0241] Example 24: Preparation of Tripterygium wilfordii liposomes

[0242] Weigh out 0.2g of triptolide lignocerate, 3g of sphingomyelin, and 0.15g of DSPE-PEG2000 according to the prescribed dosage, and dissolve them in 4g of anhydrous ethanol. Heat at 45℃ to obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 45℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Add an equal amount of water for injection and ultrafilter four times to remove the organic solvent. Make up the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0243] The average particle size was measured to be 96.6 nm, and the PDI was 0.138.

[0244] Example 25: Preparation of Tripterygium wilfordii liposomes

[0245] Weigh 0.3g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription and place them in 3g of anhydrous ethanol. Heat at 60℃ to dissolve and obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Make up to 100mL with water for injection, filter through a 0.22μm filter membrane for sterilization, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0246] The average particle size was measured to be 102.1 nm, and the PDI was 0.137.

[0247] Example 26: Preparation of Tripterygium wilfordii liposomes

[0248] Weigh 0.7g of triptolide lignocerate, 5g of soybean lecithin, 4g of phosphatidylethanolamine, and 0.15g of DSPE-PEG2000 according to the prescription and place them in 11g of anhydrous ethanol. Heat at 50℃ to dissolve and obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid and add an equal amount of water for injection for ultrafiltration 6 times to remove the organic solvent and obtain the liposome solution. Weigh 500μg of NaCl into the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Filter through a 0.22μm filter membrane for sterilization, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0249] The average particle size was measured to be 120.3 nm, and the PDI was 0.115.

[0250] Example 27: Preparation of Tripterygium wilfordii liposomes

[0251] Weigh 0.5g of triptolide lignocerate, 6g of high-purity egg yolk lecithin, and 0.3g of DSPE-PEG2000 according to the prescription and place them in 6g of anhydrous ethanol. Heat at 55℃ to dissolve and obtain the organic phase. Weigh 100μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 55℃ to dissolve and obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain the crude liposome product. Homogenize the crude liposome product twice each at 15000psi, 20000psi, and 25000psi using a high-pressure homogenizer. Collect the liposome liquid and add an equal amount of water for injection with the same electrolyte concentration as the water above. Ultrafilter four times to remove the organic solvent and obtain the liposome solution. Make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain the triptolide lignocerate liposome injection solution.

[0252] The average particle size was measured to be 109.1 nm, and the PDI was 0.102.

[0253] Example 28: Preparation of Tripterygium wilfordii liposomes

[0254] Weigh out 0.4g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription, and dissolve them in 8g of anhydrous ethanol. Heat at 50℃ to obtain the organic phase. Weigh out 20μg of NaCl and dissolve it in an appropriate amount of water for injection. Heat and stir at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, collect the liposome liquid, add an equal amount of water for injection with the same electrolyte concentration as the above water, and ultrafilter five times to remove the organic solvent to obtain the liposome solution. Make up the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0255] The average particle size was measured to be 100.1 nm, and the PDI was 0.101.

[0256] Example 29: Preparation of Tripterygium wilfordii liposomes

[0257] Weigh out 0.3g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription, and dissolve them in 4g of anhydrous ethanol. Heat at 60℃ to obtain the organic phase. Weigh out 50μg of NaCl and dissolve it in an appropriate amount of water for injection. Heat and stir at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, and collect the liposome liquid. Add an equal amount of water for injection with the same electrolyte concentration as the above water and ultrafilter four times to remove the organic solvent to obtain the liposome solution. Make up the volume with water for injection to 100mL. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0258] The average particle size was measured to be 87.2 nm, and the PDI was 0.092.

[0259] Example 30: Preparation of Tripterygium wilfordii liposomes

[0260] Weigh 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription and place them in 3g of anhydrous ethanol. Heat at 40℃ to dissolve and obtain the organic phase. Weigh 10μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 40℃ to dissolve and obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid and add an equal amount of water for injection with the same electrolyte concentration as the above water. Ultrafilter four times to remove organic solvents to obtain liposome solution. Make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0261] The average particle size was measured to be 82.7 nm, and the PDI was 0.098.

[0262] Example 31: Preparation of Tripterygium wilfordii liposomes

[0263] Weigh out 0.2g of triptolide lignocerate, 2g of hydrogenated soybean lecithin, 1g of dipalmitoylphosphatidylcholine, and 0.3g of DSPE-PEG2000 as prescribed, and place them in 2g of anhydrous ethanol. Heat at 55℃ to dissolve, obtaining the organic phase. Weigh out an appropriate amount of water for injection and heat at 55℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, to obtain liposome solutions. Weigh 20g of sucrose into the above liposome solutions, stir to dissolve, and dilute to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain lyophilized triptolide lignocerate liposome powder for injection.

[0264] The average particle size was measured to be 85.0 nm, and the PDI was 0.139.

[0265] Example 32: Preparation of Tripterygium wilfordii liposomes

[0266] Weigh 1g of triptolide lignocerate, 7g of high-purity egg yolk lecithin, and 3g of phosphatidylserine according to the prescription, and dissolve them in 15g of anhydrous ethanol at 50℃ to obtain the organic phase. Weigh an appropriate amount of water for injection and heat it at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter 7 times to remove the organic solvent to obtain the liposome solution. Weigh 40g of trehalose and place it in the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0267] The average particle size was measured to be 138.9 nm, and the PDI was 0.175.

[0268] Example 33: Preparation of Tripterygium wilfordii liposomes

[0269] Weigh 0.5g of triptolide lignocerate, 5g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription, and place them in 5g of anhydrous ethanol. Heat at 45℃ to dissolve them to obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 45℃ to obtain the aqueous phase. Slowly inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter four times to remove the organic solvent to obtain the liposome solution. Weigh 15g of sucrose and place it in the above liposome solution. Stir to dissolve it and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0270] The average particle size was measured to be 125.8 nm, and the PDI was 0.151.

[0271] Example 34: Preparation of Tripterygium wilfordii liposomes

[0272] Weigh out 0.1g of triptolide lignocerate, 0.7g of sphingomyelin, 0.3g of phosphatidylcholine, and 0.15g of DSPE-PEG2000 according to the prescription amount, and place them in 0.1g of anhydrous ethanol. Heat at 25℃ to dissolve them and obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 25℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.05μm respectively to obtain liposome solutions. Weigh out 20g of sucrose and place it in the above liposome solutions. Stir to dissolve it and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0273] The average particle size was measured to be 70.6 nm, and the PDI was 0.152.

[0274] Example 35: Preparation of Tripterygium wilfordii liposomes

[0275] Weigh out 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.25g of DSPE-PEG2000 according to the prescription, and place them in 3g of anhydrous ethanol. Heat at 45℃ to dissolve them to obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 45℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.05μm to obtain liposome solutions. Weigh out 20g of sucrose and place it in the above liposome solutions. Stir to dissolve it and make up to 100mL with water for injection. Filter through a 0.22μm filter membrane for sterilization, dispense, freeze-dry, and cap to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0276] The average particle size was measured to be 101.4 nm, and the PDI was 0.136.

[0277] Example 36: Preparation of Tripterygium wilfordii liposomes

[0278] Weigh out 3g of triptolide lignocerate, 15g of soybean lecithin, and 1g of DSPE-PEG2000 according to the prescription, and place them in 15g of anhydrous ethanol. Heat at 50℃ to dissolve them, obtaining the organic phase. Weigh out an appropriate amount of water for injection and heat at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.1μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter 7 times to remove the organic solvent to obtain the liposome solution. Weigh out 20g of maltose and 10g of trehalose, place them in the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0279] The average particle size was measured to be 148.8 nm, and the PDI was 0.194.

[0280] Example 37: Preparation of Tripterygium wilfordii liposomes

[0281] Weigh out 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.5g of DSPE-PEG2000 according to the prescription, and place them in 3g of anhydrous ethanol. Heat at 55℃ to dissolve them to obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 55℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Homogenize the crude liposomes twice under high pressure at 15000psi, 20000psi, and 25000psi respectively to obtain liposome solutions. Weigh 25g of sucrose into the above liposome solution and make up to 100mL with water for injection. Filter through a 0.22μm filter membrane for sterilization, dispense, freeze-dry, and cap to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0282] The average particle size was measured to be 106.1 nm, and the PDI was 0.141.

[0283] Example 38: Preparation of Tripterygium wilfordii liposomes

[0284] Weigh 0.4g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription, place them in 5g of anhydrous ethanol, and heat at 60℃ to dissolve them to obtain the organic phase; weigh an appropriate amount of water for injection, heat at 60℃ to obtain the aqueous phase; slowly inject the aqueous phase into the organic phase under stirring, mix well, and obtain crude liposomes; extrude the crude liposomes through extrusion membranes with pore sizes of 0.2μm, 0.1μm, and 0.05μm respectively, collect the liposome liquid, add an equal amount of water for injection, ultrafilter four times to remove organic solvent, and obtain liposome solution; weigh 10g of sucrose and 10g of trehalose into the above liposome solution, and make up to 100mL with water for injection; filter through a 0.22μm filter membrane for sterilization, dispense, freeze dry, and cap to obtain triptolide lignocerate liposome lyophilized powder for injection.

[0285] The average particle size was measured to be 115.8 nm, and the PDI was 0.149.

[0286] Example 39: Preparation of Tripterygium wilfordii liposomes

[0287] Weigh out 0.1g of triptolide lignocerate, 2g of hydrogenated soybean lecithin, and 0.5g of DSPE-PEG2000 according to the prescription, place them in 5g of tert-butanol, and heat at 60℃ to dissolve them to obtain the organic phase; weigh out an appropriate amount of water for injection, and heat at 60℃ to obtain the aqueous phase; slowly inject the aqueous phase into the organic phase under stirring, mix well, and obtain crude liposomes; extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.1μm respectively to obtain liposome solutions; weigh out 20g of maltose and 10g of sucrose into the above liposome solutions, stir until dissolved, and make up to 100mL with water for injection, filter through a 0.22μm filter membrane for sterilization, dispense, freeze dry, and cap to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0288] The average particle size was measured to be 76.6 nm, and the PDI was 0.142.

[0289] Example 40: Preparation of Tripterygium wilfordii liposomes

[0290] Weigh 0.3g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription, place them in 5g of propylene glycol, and heat at 45℃ to dissolve them to obtain the organic phase; weigh an appropriate amount of water for injection, heat at 45℃ to obtain the aqueous phase; slowly inject the aqueous phase into the organic phase under stirring, mix well, and obtain crude liposomes; extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.05μm respectively, collect the liposome liquid, add an equal amount of water for injection, ultrafilter four times to remove organic solvent, and obtain liposome solution; weigh 20g of trehalose, place it in the above liposome solution, stir until dissolved, and make up to 100mL with water for injection; filter through a 0.22μm filter membrane for sterilization, dispense, freeze dry, and cap to obtain triptolide lignocerate liposome lyophilized powder for injection.

[0291] The average particle size was measured to be 110.5 nm, and the PDI was 0.155.

[0292] Example 41: Preparation of Tripterygium wilfordii liposomes

[0293] Weigh out 0.9g of triptolide creosote, 6g of high-purity egg yolk lecithin, 4g of phosphatidylcholine, and 0.15g of DSPE-PEG2000 as prescribed, place them in 10g of anhydrous ethanol, and heat at 50℃ to dissolve them, obtaining the organic phase; weigh out NaCl... 50 μg was placed in an appropriate amount of water for injection and heated and stirred at 50 °C to dissolve, obtaining an aqueous phase. Under stirring, the aqueous phase was slowly injected into the organic phase and mixed to obtain crude liposomes. The crude liposomes were homogenized three times each at 15000 psi, 20000 psi, and 25000 psi using a high-pressure homogenizer. The liposome liquid was collected, and an equal amount of water for injection with the same electrolyte concentration as the water was added. The mixture was ultrafiltered five times to remove the organic solvent, obtaining a liposome solution. 30 g of maltose was weighed into the above liposome solution and diluted to 100 mL with water for injection. The solution was then filtered through a 0.22 μm filter membrane for sterilization, dispensed, lyophilized, and capped to obtain the lyophilized powder for injection of triptolide lignocerin liposomes.

[0294] The average particle size was measured to be 140.4 nm, and the PDI was 0.167.

[0295] Example 42: Preparation of Tripterygium wilfordii liposomes

[0296] Weigh out 0.4g of triptolide oxyacetate, 4g of high-purity egg yolk lecithin, and 0.2g of DSPE-PEG2000 according to the prescription, and place them in 6g of anhydrous ethanol. Heat at 50℃ to dissolve them, obtaining the organic phase. Weigh out 50μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 50℃ to dissolve it, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid, add an equal amount of water for injection with the same electrolyte concentration as the above water, and ultrafilter four times to remove the organic solvent, obtaining the liposome solution. Weigh 30g of sucrose and place it in the above liposome solution. Stir to dissolve and dilute to 100mL with water for injection. Sterilize by passing through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerin ester liposome.

[0297] The average particle size was measured to be 96.8 nm, and the PDI was 0.089.

[0298] Example 43: Preparation of Tripterygium wilfordii liposomes

[0299] Weigh out 0.5g of triptolide creosote, 2g of sphingomyelin, 3g of phosphatidylcholine, and 0.2g of DSPE-PEG2000 according to the prescription, and place them in 10g of anhydrous ethanol. Heat at 45℃ to dissolve them, obtaining the organic phase. Weigh out 500μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 35℃ to dissolve it, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid, add an equal amount of water for injection with the same electrolyte concentration as the above water, and ultrafilter five times to remove the organic solvent, obtaining the liposome solution. Weigh 25g of sucrose and place it in the above liposome solution. Stir to dissolve and dilute to 100mL with water for injection. Sterilize by passing through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerin ester liposome.

[0300] The average particle size was measured to be 115.2 nm, and the PDI was 0.098.

[0301] Example 44: Preparation of Tripterygium wilfordii liposomes

[0302] Weigh 0.3g of triptolide lignocerate and 5g of high-purity egg yolk lecithin according to the prescription, place them in 5g of propylene glycol, and heat at 40℃ to dissolve, obtaining the organic phase; weigh 100μg of NaCl and place it in an appropriate amount of water for injection, heat and stir at 40℃ to dissolve, obtaining the aqueous phase; under stirring, slowly inject the aqueous phase into the organic phase and mix well to obtain crude liposomes; extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm respectively, collect the liposome liquid, add an equal volume of water for injection with the same electrolyte concentration as the above water, ultrafilter four times to remove organic solvent, and obtain liposome solution. Weigh 20g of trehalose and place it in the above liposome solution, stir to dissolve, and make up to 100mL with water for injection, filter through a 0.22μm filter membrane for sterilization, dispense, lyophilize, and cap to obtain lyophilized powder for injection of triptolide lignocerate liposomes.

[0303] The average particle size was measured to be 86.1 nm, and the PDI was 0.087.

[0304] Example 45: Preparation of Tripterygium wilfordii liposomes

[0305] Weigh out 0.1g of triptolide lignocerate, 1g of dipalmitoylphosphatidylcholine, and 0.5g of DSPE-PEG2000 according to the prescription, and dissolve them in 1g of anhydrous ethanol. Heat at 30℃ to obtain the organic phase. Weigh out an appropriate amount of water for injection and heat at 30℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Homogenize the crude liposomes twice each at 15000psi, 20000psi, and 25000psi using a high-pressure homogenizer to obtain liposome solutions. Weigh out 500μg of Na2SO4 and 30g of maltose and add them to the above liposome solution. Stir to dissolve and bring the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, lyophilize, and cap to obtain lyophilized powder for injection of triptolide lignocerate liposomes.

[0306] The average particle size was measured to be 65.1 nm, and the PDI was 0.087.

[0307] Example 46: Preparation of Tripterygium wilfordii liposomes

[0308] Weigh 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.01g of DSPE-PEG2000 according to the prescription, and dissolve them in 2g of anhydrous ethanol at 55℃ to obtain the organic phase. Weigh 50μg of NaCl and dissolve it in an appropriate amount of water for injection at 55℃ with stirring to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Weigh 25g of sucrose and add it to the above liposome solutions, stir to dissolve, and bring the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0309] The average particle size was measured to be 81.3 nm, and the PDI was 0.076.

[0310] Example 47: Preparation of Tripterygium wilfordii liposomes

[0311] Weigh 0.2g of triptolide lignocerate, 4g of high-purity egg yolk lecithin, and 0.5g of DSPE-PEG2000 according to the prescription, and place them in 6g of tert-butanol. Heat at 50℃ to dissolve, obtaining the organic phase. Weigh an appropriate amount of water for injection and heat at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Weigh 60μg of NaCl, 40μg of Na2SO4, and 20g of maltose into the above liposome solutions, stir to dissolve, and dilute to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, lyophilize, and cap to obtain lyophilized powder for injection of triptolide lignocerate liposomes.

[0312] The average particle size was measured to be 79.1 nm, and the PDI was 0.083.

[0313] Example 48: Preparation of Tripterygium wilfordii liposomes

[0314] Weigh 0.1g of triptolide lignocerate and 1g of high-purity egg yolk lecithin according to the prescription, and dissolve them in 0.1g of anhydrous ethanol. Heat at 30℃ to obtain the organic phase. Weigh 500μg of Na3PO4 and dissolve it in an appropriate amount of water for injection. Heat and stir at 30℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm to obtain liposome solutions. Weigh 12g of sucrose and add it to the above liposome solutions. Stir to dissolve, and bring the volume to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0315] The average particle size was measured to be 63.2 nm, and the PDI was 0.043.

[0316] Example 49: Preparation of Tripterygium wilfordii liposomes

[0317] Weigh out 0.4g of triptolide oxyacetate, 5g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription, and place them in 4g of anhydrous ethanol. Heat at 55℃ to dissolve them, obtaining the organic phase. Weigh out 10μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 55℃ to dissolve it, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid, add an equal amount of water for injection with the same electrolyte concentration as the above water, and ultrafilter four times to remove the organic solvent, obtaining the liposome solution. Weigh 12g of sucrose and 8g of trehalose into the above liposome solution, stir to dissolve, and dilute to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerin ester liposome.

[0318] The average particle size was measured to be 95.5 nm, and the PDI was 0.094.

[0319] Example 50: Preparation of Tripterygium wilfordii liposomes

[0320] Weigh 0.5g of triptolide lignocerate, 6g of high-purity egg yolk lecithin, and 0.4g of DSPE-PEG2000 according to the prescription, and place them in 8g of propylene glycol. Heat at 60℃ to dissolve, obtaining the organic phase. Weigh 50μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 60℃ to dissolve, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively. Collect the liposome liquid, add an equal volume of water for injection with the same electrolyte concentration as the above water, and ultrafilter five times to remove the organic solvent, obtaining the liposome solution. Weigh 15g of sucrose and place it in the above liposome solution. Stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, lyophilize, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0321] The average particle size was measured to be 112.0 nm, and the PDI was 0.126.

[0322] Example 51: Preparation of Tripterygium wilfordii liposomes

[0323] Weigh out 0.1g of triptolide lignocerate, 1g of high-purity egg yolk lecithin, and 0.3g of DSPE-PEG2000 according to the prescribed amount, and dissolve them in 2g of anhydrous ethanol by heating at 50℃ to obtain the organic phase. Weigh out 80μg of NaCl, 12g of maltose, and 13g of trehalose, and dissolve them in an appropriate amount of water for injection by heating and stirring at 50℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain the crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm respectively to obtain liposome solutions. Make up to 100mL with water for injection; filter through a 0.22μm filter membrane for sterilization, dispense, lyophilize, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0324] The average particle size was measured to be 75.5 nm, and the PDI was 0.087.

[0325] Example 52: Preparation of Tripterygium wilfordii liposomes

[0326] Weigh out 0.4g of triptolide creosote, 5g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription, and place them in 8g of anhydrous ethanol. Heat at 55℃ to dissolve, obtaining the organic phase. Weigh out 60μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 55℃ to dissolve, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.05μm, respectively. Collect the liposome liquid, add an equal amount of water for injection with the same electrolyte concentration as the above water, and ultrafilter five times to remove the organic solvent, obtaining the liposome solution. Weigh 25g of sucrose and place it in the above liposome solution. Stir to dissolve and dilute to 100mL with water for injection. Sterilize by passing through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerin ester liposome.

[0327] The average particle size was measured to be 96.6 nm, and the PDI was 0.148.

[0328] Example 53: Preparation of Tripterygium wilfordii liposomes

[0329] Weigh 0.5g of triptolide lignocerate, 5g of high-purity egg yolk lecithin, and 0.2g of DSPE-PEG2000 according to the prescribed dosage, and dissolve them in 5g of anhydrous ethanol by heating at 55℃ to obtain the organic phase. Weigh 10μg of NaCl and dissolve it in an appropriate amount of water for injection by heating and stirring at 55℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter four times to remove the organic solvent to obtain the liposome solution. Weigh 20g of sucrose into the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, lyophilize, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0330] The average particle size was measured to be 120.4 nm, and the PDI was 0.125.

[0331] Example 54: Preparation of Tripterygium wilfordii liposomes

[0332] Weigh 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.25g of DSPE-PEG2000 according to the prescription, and dissolve them in 3g of propylene glycol. Heat at 45℃ to obtain the organic phase. Weigh 70μg of NaCl and dissolve it in an appropriate amount of water for injection at 45℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.1μm, and 0.05μm, respectively, collect the liposome liquid, add an equal volume of water for injection with the same electrolyte concentration as the above water, and ultrafilter four times to remove the organic solvent to obtain the liposome solution. Weigh 30g of sucrose into the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0333] The average particle size was measured to be 85.5 nm, and the PDI was 0.096.

[0334] Example 55: In vivo antitumor effect of triptolide lignocerin ester liposomes

[0335] Based on the triptolide lignocerin ester liposomes prepared in Examples 35, 42, and 53, an antitumor study was conducted on a Panc 02 pancreatic cancer model, using TP (DMSO) solution and Minnelide solution as controls. The experimental design and results are as follows:

[0336] 1. Sample Source

[0337] TP (DMSO) solution and Minnelide solution were used as positive control drugs; TP-LA liposomes prepared in Examples 35, 42 and 53 were used as liposome formulations.

[0338] 2. Establishment of a mouse Panc 02 pancreatic cancer tumor model and design of a drug administration regimen

[0339] Mouse pancreatic cancer Panc 02 cells were cultured in DMEM medium at 37°C under standard conditions of 5% CO2, and passaged on average every two days. At the logarithmic growth phase, the cell concentration was adjusted to 1×10⁻⁶ cells using culture medium. 7 Panc02 cells were subcutaneously inoculated into the right axilla of ICR mice under aseptic conditions at a rate of 100-300 mm² / mL to establish a mouse pancreatic cancer model. 3Mice were randomly divided into 6 groups (n=6 per group) based on tumor volume. These groups included a control group, a TP (DMSO) solution group, a Minnelide solution group, and a TP-LA liposome dosage group. Mice were administered the drug via tail vein injection at a dose of 0.6 mg / kg (based on TP). The control group received 0.2 mL of physiological saline. The drugs were administered every two days for a total of four doses. Mice were sacrificed on the third day after drug withdrawal, and their weight, tumor removal, and weighing were performed to calculate the tumor inhibition rate.

[0340]

[0341] 3. Anti-tumor effects

[0342] Using mouse pancreatic cancer as a model, the antitumor effects of triptolide creosote ester liposomes, Minnelide solution, and TP (DMSO) solution were investigated. The results are shown in Table 17.

[0343] Table 17 Comparison of the antitumor effects of Minnelide and Tripterygium wilfordii liposomes

[0344]

[0345] Note: Compared to the model group * P < 0.05 ** P < 0.01; compared with the TP group, # P < 0.05 ## P < 0.01.

[0346] 4. Results Analysis

[0347] TP-LA liposomes prepared in Examples 35, 42, and 53 were used to evaluate the pharmacodynamics of a mouse Panc 02 pancreatic cancer model in combination with TP solution and Minnelide solution. The experimental results showed ( Figure 3 The prepared TP-LA liposomes showed significantly better therapeutic effects than TP solution and Minnelide solution. Mice administered TP solution experienced a significant decrease in body weight, while mice administered Minnelide showed greater individual variation in body weight, with a significantly different overall growth trend compared to the negative control group. Furthermore, intravenous injection of TP solution and Minnelide solution caused varying degrees of tail swelling and ulceration in mice, producing significant irritation. Figure 4 Mice treated with TP-LA liposomes showed no significant irritation and exhibited a normal weight gain trend, indicating that the liposomes prepared in this invention have significantly improved overall efficacy and biosafety.

[0348] Example 56: Irritation evaluation of the injectable triptolide lignocerin ester liposomes prepared in Examples 35, 42, and 53.

[0349] 1. Stimulus program design

[0350] Eighteen rabbits (weighing 1.5-2.0 kg) were randomly divided into 6 groups. Each group received an injection of saline, TP-LA liposomes prepared in Examples 35, 42, and 53, TP (DMSO) solution, or Minnelide solution via the marginal ear vein in the right ear. The dosage was 0.22 mg / kg (based on TP), and the injection rate was 1 mL / min. Simultaneously, an equal volume of saline was administered via the marginal ear vein in the left ear as a control. This treatment was repeated once daily for three consecutive days.

[0351] Animal behavior and pathological changes at the injection site were observed visually during daily administration. Euthanasia was performed 48 hours after the last administration. Auricular vein tissue was harvested approximately 2.0 cm centripetally from the injection site, fixed in 10% paraformaldehyde solution, dehydrated using an ethanol gradient, embedded in paraffin, and stained with hematoxylin and ovoglobulin. All samples were examined using a BX43-DP21 light microscope (Olympus Corporation, Japan), and pathological changes were assessed.

[0352] 2. The analysis of the stimuli evaluation results is as follows:

[0353] like Figure 5 As shown, in the control group, the marginal ear veins of rabbits showed no obvious congestion or bleeding, the vessel walls were intact, and there were no obvious inflammatory lesions in the surrounding tissues. The stratified squamous epithelium on the surface of the auricle skin was intact, and there were no obvious lesions in the subcutaneous hair follicles, sebaceous glands, and sweat glands. The elastic cartilage in the central area was normal in morphology, and the interstitial fibrous tissue of the auricle showed no obvious lesions, although there was a small amount of chronic inflammatory cell infiltration in the interstitium.

[0354] The TP (DMSO) solution group showed significant bleeding, accompanied by necrosis of the vessel walls and marked inflammatory cell infiltration in the surrounding tissues. Subcutaneous connective tissue was loose and edematous, with broken collagen fibers; acute inflammatory cell infiltration was observed in the interstitial fibrous tissue of the auricle. The Minnlide solution group showed ecchymosis with some bleeding. Surrounding tissues showed edema and slight leakage of tissue fluid.

[0355] The results of the liposomes containing triptolide oxytocin for injection (Examples 35, 42, and 53) were largely consistent with the control group. The endothelial cells of the marginal ear veins were essentially intact, with only a small amount of inflammatory cell infiltration around the vessel walls. No obvious degeneration, necrosis, or hyperplasia was observed in the epidermal tissue. The elastic cartilage in the central area was morphologically normal, with normal chondrocytes, cartilage matrix, and perichondrium structure visible.

[0356] Example 57: Preparation of Tripterygium wilfordii liposomes

[0357] Weigh 0.3g of triptolide lignocerate and 4g of high-purity egg yolk lecithin according to the prescription and place them in 5g of anhydrous ethanol. Heat and stir at 60℃ to dissolve them to obtain the organic phase. Weigh an appropriate amount of water for injection and heat at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.1μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter four times to remove the organic solvent to obtain the liposome solution. Make up the volume with water for injection to 100mL. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0358] The average particle size was measured to be 100.6 nm, and the PDI was 0.104.

[0359] Example 58: Preparation of Tripterygium wilfordii liposomes

[0360] Weigh 0.3g of triptolide lignocerate, 3g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription and place them in 3g of anhydrous ethanol. Heat and stir at 55℃ to dissolve, obtaining the organic phase. Weigh 10μg of NaCl and place it in an appropriate amount of water for injection. Heat and stir at 55℃ to dissolve, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.2μm, 0.1μm, and 0.05μm respectively to obtain liposome solutions. Make up the volume with water for injection to 100mL. Sterilize by filtering through a 0.22μm filter membrane, dispense, and cap to obtain triptolide lignocerate liposome injection solution.

[0361] The average particle size was measured to be 79.2 nm, and the PDI was 0.092.

[0362] Example 59: Preparation of Tripterygium wilfordii liposomes

[0363] Weigh 0.4g of triptolide lignocerate, 5g of high-purity egg yolk lecithin, and 0.1g of DSPE-PEG2000 according to the prescription and place them in 9g of anhydrous ethanol. Heat and stir at 60℃ to dissolve, obtaining the organic phase. Weigh an appropriate amount of water for injection and heat at 60℃ to obtain the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.1μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter 5 times to remove the organic solvent, obtaining the liposome solution. Weigh 25g of sucrose into the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder injection of triptolide lignocerate liposomes.

[0364] The average particle size was measured to be 108.5 nm, and the PDI was 0.147.

[0365] Example 60: Preparation of Tripterygium wilfordii liposomes

[0366] Weigh out 0.3g of triptolide lignocerate, 3.7g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescription, and place them in 5g of anhydrous ethanol. Heat and stir at 55℃ to dissolve, obtaining the organic phase. Weigh out an appropriate amount of water for injection, and heat and stir at 55℃ to dissolve, obtaining the aqueous phase. Slowly inject the aqueous phase into the organic phase under stirring and mix well to obtain crude liposomes. Extrude the crude liposomes through extrusion membranes with pore sizes of 0.4μm, 0.2μm, and 0.1μm, respectively, collect the liposome liquid, add an equal amount of water for injection, and ultrafilter four times to remove the organic solvent, obtaining the liposome solution. Weigh 20g of sucrose into the above liposome solution, stir to dissolve, and make up to 100mL with water for injection. Sterilize by filtering through a 0.22μm filter membrane, dispense, freeze-dry, and cap to obtain the lyophilized powder for injection of triptolide lignocerate liposomes.

[0367] The average particle size was measured to be 105.5 nm, and the PDI was 0.112.

[0368] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A triptolide oxyphylla ester, the chemical structure of which is shown in formula (Ⅰ): Formula (I).

2. The method for preparing triptolide oxyphylla ester as described in claim 1, characterized in that, The triptolide lignocerate ester is obtained by esterification of triptolide and lignoceric acid. The synthetic route is as follows: The preparation method is as follows: weigh out lignoceric acid and acid-binding agent and dissolve them in anhydrous DCM, and stir at room temperature to obtain a mixed solution; weigh out the catalyst and dissolve it in the above mixed solution, and stir to mix evenly; slowly add triptolide dissolved in anhydrous DCM to the above mixed solution, and continue the reaction under oil bath heating and nitrogen protection conditions; wash the reaction solution twice with saturated NaHCO3 solution and saturated NaCl solution respectively, and remove residual water with anhydrous Na2SO4 after separation, then remove DCM by rotary evaporation, and finally separate and purify by silica gel column chromatography to obtain triptolide lignoceric acid ester.

3. The preparation method according to claim 2, characterized in that, The acid-binding agent is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, p-nitrobenzoyl chloride, and N,N'-diisopropylcarbodiimide; the catalyst is one or more of 4-pyrrolidinylpyridine, 4-dimethylaminopyridine, N,N-diisopropylethylamine, and 1-hydroxybenzotriazole.

4. The preparation method according to claim 2, characterized in that, The molar ratio of lignoceric acid to triptolide is 2:1 to 6:1; the molar ratio of the acid-binding agent to triptolide is 2:1 to 6:

1.

5. The preparation method according to claim 2, characterized in that, The oil bath temperature is 25 ~ 80 ℃; the reaction time is 8 ~ 24 h.

6. A nano-formulation of triptolide oxyphylla ester as described in claim 1, characterized in that, The nano-formulation is triptolide lignocerin ester liposome.

7. The nano-formulation according to claim 6, characterized in that, The triptolide oxyphylla liposome is an injection solution or a lyophilized powder for injection.

8. The nano-formulation according to claim 6, characterized in that, The triptolide lignocerate liposomes described herein contain triptolide lignocerate as the main drug and also include lecithin.

9. The nano-formulation according to claim 6, characterized in that, The triptolide oxycetetracycline liposomes, with a drug-to-lipid ratio of 1:5-1:30 (w / w), are formulated from the following ingredients:

10. The nano-formulation according to claim 9, characterized in that, The triptolide oxycetetracycline liposomes, with a drug-to-lipid ratio of 1:10-1:20 (w / w), are formulated from the following composition:

11. The nanoformulation according to claim 9 or 10, characterized in that, The lecithin is selected from one or more of the following: high-purity egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, phosphatidylcholine, soybean lecithin, phosphatidylserine, myristoylphosphatidylcholine, distearate phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; the organic solvent is selected from one or more of the following: anhydrous ethanol, propylene glycol, tert-butanol, chloroform, and dichloromethane; the electrolyte is selected from one or more of the following: sodium citrate, Na2SO4, NaCl, Na2CO3, FeCl3, and Na3PO4; and the freeze-drying protectant is selected from one or more of the following: trehalose, sucrose, maltose, lactose, mannitol, glucose, sorbitol, xylitol, erythritol, and threonine.

12. A method for preparing triptolide lignan ester liposomes, characterized in that, The formulation of the triptolide lignocerin liposomes is as described in claim 9 or 10, and the preparation method is as described in claim 10. The process includes the following steps: Weigh the prescribed amounts of triptolide lignocerate, lecithin, and DSPE-PEG2000, place them in an organic solvent for injection, and heat at 25–60 °C to dissolve them, obtaining the organic phase; Weigh the prescribed amounts of electrolyte into an appropriate amount of water for injection, and heat and stir at 25–60 °C to dissolve them, obtaining the aqueous phase; Under stirring conditions, slowly inject the aqueous phase into the organic phase or slowly inject the organic phase into the aqueous phase, mix well, and obtain crude liposomes; Emulsify the crude liposomes, which can be done by homogenizing and emulsifying them in a high-pressure homogenizer, or by extruding them sequentially through extrusion membranes of different pore sizes in an extruder, or by high-pressure homogenization followed by extrusion, to obtain a liposome solution; Make up to the total volume with water for injection, filter through a 0.22 μm filter membrane for sterilization, dispense, seal, and cap, to obtain triptolide lignocerate liposome injection solution; or Weigh the prescribed amount of lyophilization protectant, place it in the liposome liquid, stir to dissolve it, and make up to the total volume with water for injection; Filter through a 0.22 μm filter membrane for sterilization, dispense, seal, and cap, to obtain triptolide lignocerate liposome injection solution; or Weigh the prescribed amount of lyophilization protectant, place it in the liposome liquid, stir to dissolve it, and make up to the total volume with water for injection; Filter through a 0.22 μm filter membrane for sterilization, dispense, seal, and cap, to obtain triptolide lignocerate liposome injection solution. Sterilization is achieved through filtration using a μm filter membrane, followed by dispensing, freeze-drying, and sealing to obtain the lyophilized powder injection of triptolide lignocerin ester liposomes.

13. The preparation method according to claim 12, characterized in that, The pore size of the extruded membrane is selected from one or more of 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1 μm, and 0.05 μm, and during extrusion, it passes through the pores from the larger pore size to the smaller pore size in sequence.

14. The use of the triptolide oxycarboxylate ester as described in claim 1, and the nanoformation of triptolide oxycarboxylate ester as described in any one of claims 6-11, in the preparation of antitumor drugs.

Images