Norcantharidin liposome injection as well as preparation method and application thereof

Abstract

The invention discloses a norcantharidin liposome injection as well as a preparation method and application thereof. The injection is prepared from the following components in parts by mass: 5 to 15 parts of norcantharidin, 50 to 200 parts of phospholipid, 30 to 40 parts of DSPE-mPEG2000, 25 to 50 parts of cholesterol and 79.1 to 118.6 parts of calcium acetate. The blank liposome is prepared through a film hydration method, the encapsulation efficiency of the preparation is improved through a calcium acetate active drug loading method, the prepared preparation can remarkably enhance the anti-tumor effect of the preparation by improving the targeting property of the drug and prolonging the in-vivo action time of the drug, irritation of the drug to blood vessels and damage of the drug to other organs are reduced, and the anti-tumor effect of the preparation is improved. And the purposes of effect enhancement and toxicity reduction are achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to a norcantharidin liposome injection, a preparation method and an application thereof. Background Art

[0002] According to the 2020 Global Cancer Statistics Report, there are an estimated 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020, of which liver cancer is the third leading cause of death. Liver cancer, represented by hepatocellular carcinoma, is the sixth most common cancer in the world. It has the characteristics of high mortality, short survival (median survival is only 11 months), low early diagnosis rate, rapid disease progression, high malignancy, and poor prognosis, which has caused a serious burden on global health. In particular, in China, the incidence of hepatocellular carcinoma (HCC) is as high as 35 / 100,000, accounting for nearly half of the global HCC treatment burden. There is still a long way to go in the prevention and treatment of HCC.

[0003] Norcantharidin (NCTD) is a demethylated derivative of cantharidin (CTD). It has unique advantages in the treatment of tumors and is the preferred auxiliary drug for the treatment of various digestive tract cancers such as liver cancer, esophageal cancer, gastric cancer and cardiac cancer, among which primary liver cancer is the main one. NCTD can not only play a role in cell apoptosis and inhibit cell secretion of inflammatory factors by downregulating the activation level of the NF-κB signaling pathway, but also inhibit tumor cell proliferation, increase white blood cells, regulate immune response and do not produce bone marrow suppression. In addition, NCTD can also improve liver function and is used to treat hepatitis, cirrhosis and hepatitis B virus. In summary, as a representative potent anti-tumor ingredient in traditional Chinese medicine, norcantharidin has multiple biological activities and is currently the only anti-tumor ingredient that has the effect of increasing white blood cells, and has great development value.

[0004] However, norcantharidin has poor water solubility, a short half-life, is easily cleared from the body, and is accompanied by significant nephrotoxicity and cardiotoxicity, which greatly limits its clinical application. At present, norcantharidin is mainly used in clinical practice in the form of norcantharidin tablets and injections. Although demethylation significantly reduces the toxicity of cantharidin, it does not completely eliminate it. In clinical use, some patients experience adverse reactions when taking more than 30 mg orally per day, mainly manifested as nausea, vomiting, dizziness and other symptoms. The recommended clinical usage and dosage of sodium norcantharidin injection is: intravenous injection, diluted with 5% glucose injection, slowly intravenously pushed, 10 to 30 mg at a time. Since the injection is an alkaline solution, its pH value is about 8-10, which is much higher than the blood pH value (7.35-7.45), so it has a greater irritation to the blood vessel wall and is prone to chemical phlebitis; and since the drug itself is highly irritating, it can also cause vasospasm, blood reduction, increase the concentration of sodium norcantharidate in local blood vessels, aggravate phlebitis, and if the infusion speed is greater than the blood flow rate, the incidence of phlebitis increases significantly. Therefore, when sodium norcantharidate injection is used clinically for peripheral superficial vein infusion, the proportion of superficial phlebitis is quite high, and some patients refuse to use the drug for treatment, which greatly affects the normal use of the drug in clinical practice.

[0005] In recent years, researchers are using the targeting, slow-release and other characteristics of new preparations to enhance targeted drug delivery, reduce drug distribution in the kidneys and irritation to the urinary system, and make it more effective in tumor diseases. It mainly includes preparation into microspheres, nanoparticles, liposomes and microemulsions. Patent CN101229125A prepares a microemulsion injection containing sodium norcantharidin, and its maximum drug loading is only 76μg / mL. At the same time, the preparation process is similar to that of ordinary fat emulsions, and there is no particle size description. Patent CN115054578A prepares a norcantharidin nanolipid carrier, and although its encapsulation rate is as high as 74%, the drug loading is low and it is difficult to meet the dosage requirements of clinical medication. Patent CN107753429A prepares a norcantharidin liposome with liver targeting, and its encapsulation rate is only 28%, and its stability is poor, and it needs to be freeze-dried for storage. The relevant experimental data also did not prove its liver targeting effect. Patent CN115671048A prepares a complex of norcantharidin liposome and emulsion, and the preparation process and auxiliary materials are complicated.

[0006] Therefore, although a variety of different dosage forms of norcantharidin preparations have been disclosed, these preparations still have defects such as low encapsulation rate and drug loading, complex preparation process, and no significant targeting. In addition, the above patents have not yet stated the pharmacokinetic data and beneficial efficacy data of the preparations, and the feasibility of further development and production cannot be verified. There are still many problems that need to be further improved. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned defects of the prior art, provide a norcantharidin liposome injection and a preparation method and application thereof, solve the problems of vascular irritation, nephrotoxicity, cardiotoxicity, short half-life of norcantharidin itself, and low encapsulation rate and drug loading of norcantharidin liposome, while ensuring the stability of the liposome, achieving the purpose of increasing efficacy and reducing toxicity.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] A norcantharidin liposome injection, comprising the following components in parts by weight:

[0010] 5 to 15 parts of norcantharidin, 50 to 200 parts of phospholipids, 30 to 40 parts of DSPE-mPEG2000, 25 to 50 parts of cholesterol and 79.1 to 118.6 parts of calcium acetate.

[0011] The present invention also discloses a method for preparing the above-mentioned norcantharidin liposome injection, comprising the following steps:

[0012] (1) dissolving norcantharidin in water at 60° C. to 100° C., adding alkali to adjust the pH to 9 to 11, heating at 60° C. to 80° C. for 20 min to 40 min, and then adding acid to adjust the pH to 3 to 4 to obtain pretreated norcantharidin;

[0013] (2) Phospholipids and DSPE-mPEG 2000 and cholesterol are dissolved in an organic solvent, and the organic reagent is removed by evaporation under reduced pressure to obtain a lipid film, and then calcium acetate is added for hydration and particle size treatment to obtain large unilamellar liposomes, and then the outer aqueous phase of the large unilamellar liposomes is replaced with a sodium sulfate aqueous solution with a pH of 3 to 7 to form a pH gradient inside and outside the liposome membrane to obtain blank liposomes;

[0014] (3) Mixing the blank liposomes and the pretreated norcantharidin for drug loading to obtain the norcantharidin liposome injection.

[0015] The present invention also discloses the use of the above-mentioned norcantharidin liposome injection or the norcantharidin liposome injection prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0016] Implementing the embodiments of the present invention will have the following beneficial effects:

[0017] The invention adopts a thin film hydration method to prepare blank liposomes, and a calcium acetate active drug loading method to improve the encapsulation rate of the preparation. The encapsulation rate of the prepared norcantharidin liposome injection reaches 85% to 99.3%, and the long-term stability is good, and freeze-drying storage is not required, thereby greatly simplifying the production process and reducing the production cost.

[0018] The present invention further connects lactoferrin with liposomes by a covalent bond coupling method, and the obtained lactoferrin-modified norcantharidin liposome injection has obvious liver cancer cell targeting advantages compared with commercially available sodium norcantharidin injection, and has uniform particle size, good long-term stability and plasma stability.

[0019] The results of pharmacokinetic and pharmacodynamic experiments showed that the preparation significantly reduced vascular irritation and damage to other organs during injection, prolonged the drug action time, and had significantly better anti-tumor effects than commercially available preparations, achieving the goal of increasing efficacy and reducing toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram for the preparation of norcantharidin liposome injection and lactoferrin-modified norcantharidin liposome injection.

[0021] Figure 2 The present invention is a flow chart for the preparation of norcantharidin liposome injection and lactoferrin-modified norcantharidin liposome injection.

[0022] Figure 3 Appearance pictures of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0023] Figure 4 Transmission electron micrographs of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0024] Figure 5 The particle size and potential distribution diagram of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0025] Figure 6 This is a comparison chart of X-ray diffraction of NCTD-Lips prepared in Example 9 of the present invention and NCTD-Lips-Lf and NCTD prepared in Example 10.

[0026] Figure 7 This is a graph showing the stability test results of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0027] Figure 8 This is a graph showing the hemolysis test results of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0028] Fig. 9Tissue distribution diagram of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0029] Fig.10 The drug-time curves of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention are shown.

[0030] Fig.11 Schematic diagram of the dosing regimen of NCTD-Lips prepared in Example 9 and NCTD-Lips-Lf prepared in Example 10 of the present invention.

[0031] Fig.12 This is a comparative diagram of the anti-tumor effects of NCTD-Lips prepared in Example 9 of the present invention and NCTD-Lips-Lf prepared in Example 10 and commercially available sodium norcantharinate injection.

[0032] Fig.13 This is a comparison chart of the biochemical analysis results of NCTD-Lips prepared in Example 9 of the present invention and NCTD-Lips-Lf prepared in Example 10 and commercially available sodium norcantharinate injection.

[0033] Fig.14 This is a tissue section comparison diagram of NCTD-Lips prepared in Example 9 of the present invention and NCTD-Lips-Lf prepared in Example 10 and commercially available sodium norcantharinate injection.

[0034] Fig.15 This is a comparison chart of the vascular irritation test results of NCTD-Lips-Lf prepared in Example 10 of the present invention and the commercially available sodium norcantharidin injection. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0036] In the present invention, unless otherwise stated, the selected raw materials are commercially available products known to those skilled in the art.

[0037] The present inventors have conducted in-depth research on norcantharidin preparations and found that:

[0038] The structure of liposomes is very similar to that of cell membranes, which makes them highly biocompatible and biodegradable. Therefore, liposomes can protect drugs from being degraded by enzymes before they reach the lesion site. At the same time, drugs are "hidden" inside liposomes in the form of physical encapsulation, which can improve drug stability, reduce drug toxicity, and exert better therapeutic effects. In addition, the bilayer surface formed by amphiphilic phospholipids can be structurally modified with ligands or other functional groups through physical or chemical means to make liposomes tissue-targeted, prolong the effective residence time of liposomes in the lesion site, and even achieve efficient targeted drug delivery. Therefore, this patent is committed to preparing a liposome with high encapsulation rate and good stability to meet clinical needs.

[0039] Cantharidin acid is the in vivo metabolite of norcantharidin, with a molecular formula of C8H 10 O5 has similar anti-tumor effects, and its water solubility is significantly higher than that of norcantharidin. Its terminal carboxyl group makes norcantharidin an amphoteric weakly acidic compound, which provides an effective new idea for improving the encapsulation rate of liposomes.

[0040] Lactoferrin is a non-heme iron ion binding glycoprotein with a relative molecular weight of 80 kDa. It is mainly secreted by mucosal epithelial cells and belongs to the transferrin family. The asialoglycoprotein receptor (ASGPR) is less expressed in normal cells, but overexpressed on the surface of liver cancer cells. ASGPR-mediated targeted delivery provides great hope for improving liver cancer treatment. Lactoferrin has a high affinity for ASGPR (Kd is about 80 nmol / L), indicating that lactoferrin can be used as a specific ligand of ASGPR for the treatment of liver cancer. In vitro experimental results show that transferrin-functionalized liposomes have good targeting to liver cancer SMMC-7721 cells; in vivo experimental results show that transferrin-functionalized liposomes can effectively target liver cancer and significantly inhibit the growth of nude mouse transplanted tumors. At present, various lactoferrin-based nanomaterials have been developed as ligands for lactoferrin receptors, low-density lipoprotein-related receptors and transferrin receptors to achieve brain-targeted or breast cancer-targeted delivery of nanomaterials.

[0041] Therefore, the present inventors have arrived at the present invention as follows based on these findings.

[0042] The invention discloses a norcantharidin liposome injection, comprising the following components in parts by weight: 5 to 15 parts of norcantharidin, 50 to 200 parts of phospholipids, 30 to 40 parts of DSPE-mPEG 2000 , 25 to 50 parts of cholesterol and 79.1 to 118.6 parts of calcium acetate.

[0043] Among them, the general structural formula of norcantharidin (NCTD) is:

[0044]

[0045] Specifically, the present invention first pre-treats norcantharidin (prepares it into norcantharidin acid), and utilizes its amphoteric weak acidic properties to improve the liposome encapsulation efficiency through the calcium acetate active drug loading method. In order to reduce the recognition and phagocytosis of liposomes by macrophages in the reticuloendothelial system, thereby prolonging the circulation time of the drug in the body, this study adds DSPE-mPEG to the lipid membrane material. 2000 To prepare PEGylated liposomes, cholesterol is added to enhance membrane fluidity.

[0046] In a specific embodiment, the mass ratio of phospholipid to cholesterol is 2:1 to 4:1.

[0047] In one embodiment, phospholipids and DSPE-mPEG 2000 The molar ratio of phospholipids to DSPE-mPEG is 12:1 to 10:1. 2000 The molar ratio is 23:2 to 21:2.

[0048] In a specific embodiment, the phospholipid includes one or more of S100, E80, PC-98T, DSPC and SM. Preferably, the phospholipid is DSPC with a higher phase transition temperature.

[0049] The present invention also discloses a method for preparing the above-mentioned norcantharidin liposome injection, comprising the following steps:

[0050] (1) Pre-treating norcantharidin to improve the encapsulation efficiency, specifically, dissolving norcantharidin in water at 60°C to 80°C, adding alkali to adjust the pH to 9 to 11, heating at 60°C to 80°C for 20 min to 40 min, and then adding acid to adjust the pH to 3 to 4 to obtain pre-treated norcantharidin.

[0051] (2) Preparation of blank liposomes: Phospholipids, DSPE-mPEG 2000 and cholesterol are dissolved in an organic solvent, and the organic reagent is removed by evaporation under reduced pressure to obtain a lipid film, followed by the addition of calcium acetate for hydration and size reduction to obtain large unilamellar liposomes, and then the external aqueous phase of the large unilamellar liposomes is replaced with an aqueous sodium sulfate solution with a pH of 3 to 7 to form a pH gradient inside and outside the liposome membrane to obtain blank liposomes.

[0052] In a specific embodiment, the organic solvent includes one or more of chloroform, dichloromethane and methanol. Preferably, the organic solvent is chloroform.

[0053] In a specific embodiment, step (2) specifically comprises: weighing the prescribed amount of phospholipids, DSPE-mPEG 20001. Cholesterol is dissolved in chloroform and evaporated under reduced pressure at 38℃~45℃ to obtain a lipid film. A calcium acetate aqueous solution is added to hydrate the film at 55℃~65℃ for 15min~60min. The obtained large single-layer liposomes are filtered through 0.45μm and 0.22μm filter membranes three times respectively, and the liposome particle size is finally controlled to be 100nm~200nm. The granulated liposomes are dialyzed against a sodium sulfate aqueous solution with a pH of 3-7 to remove calcium ions in the external aqueous phase, and the external liquid is switched to a dialysis medium to form a pH gradient inside and outside the liposomes, and are stored at 4℃ for later use.

[0054] In a specific embodiment, the blank liposome granulation method includes probe ultrasound method or extrusion method, preferably extrusion method, and the specific operation is as follows: the hydrated blank liposomes are repeatedly extruded 3 to 5 times using 200 nm and 80 nm PC membranes respectively.

[0055] In a specific embodiment, the pH of the aqueous phase outside the blank liposomes is preferably pH 3-5.

[0056] (3) Drug loading of norcantharidin: blank liposomes and pretreated norcantharidin were mixed for drug loading to obtain norcantharidin liposomes (NCTD-Lips).

[0057] In a specific embodiment, the drug loading temperature is 55° C. to 65° C., and the drug loading time is 20 min to 60 min.

[0058] In a specific embodiment, the drug-lipid ratio (w / w) of the norcantharidin liposome injection is 1:10 to 1:20. Preferably, the drug-lipid ratio (w / w) is 1:10 to 1:18.

[0059] In a specific embodiment, the norcantharidin liposomes are spherical, with an average particle size of 100nm to 200nm, a 90% cumulative particle size value of ≤220nm, a content of 95% to 105% of the labeled amount, and an encapsulation efficiency of 85% to 99.9%.

[0060] Furthermore, the inventors found that PEGylated long-circulating liposomes are accompanied by a "PEG dilemma", that is, their inefficient interaction with target cells. In order to solve this problem, PEGylated liposomes can be modified by selecting suitable targeting ligands to improve their specificity for liver cancer cells. At the same time, ligand-modified liposomes are conducive to improving the targeting of the preparation, reducing the distribution and damage of the drug in other tissues and improving the therapeutic effect. Therefore, the norcantharidin liposome injection of the present invention also includes the following components in mass fractions: 2.5 to 5.3 parts of DSPE-mPEG 2000 -COOH and 1.8 to 3.2 parts of lactoferrin.

[0061] Specifically, this study selected lactoferrin as a ligand, and used the high affinity (Kd of about 80 nmol / L) of lactoferrin to ASGPR highly expressed on the surface of liver cancer cells to achieve active targeting of liposomes. At the same time, this study selected DSPC with a higher phase transition temperature as the liposome membrane material, which effectively improved its long-term stability and plasma stability, and did not require freeze-drying for storage, greatly simplified the production process, and reduced the production cost of the preparation. DSPE-mPEG is added to the norcantharidin liposome injection of the present invention. 2000 -COOH is used to connect lactoferrin to liposomes.

[0062] In one embodiment, DSPE-mPEG 2000 DSPE-mPEG 2000 The molar ratio of -COOH is 7:1 to 12:1. Preferably, DSPE-mPEG 2000 DSPE-mPEG 2000 The molar ratio of -COOH is 9:1 to 10:1.

[0063] In one embodiment, lactoferrin and DSPE-PEG 2000 The molar ratio of -COOH is 1:30 to 1:50.

[0064] The present invention also discloses a method for preparing the lactoferrin-modified norcantharidin liposome injection, comprising the following steps:

[0065] (1) According to (1) to (3) in the method for preparing the norcantharidin liposome injection in the embodiment of the present invention, the norcantharidin liposome (NCTD-Lips) was prepared.

[0066] (2) Modification of lactoferrin: DSPE-mPEGylated liposomes 2000 After mixing -COOH, NHS and EDC, lactoferrin was added for incubation to allow the amino groups on lactoferrin to react with DSPE-mPEG. 2000 The carboxyl group on -COOH was covalently coupled, and then the pH of the external aqueous phase was adjusted to 7.4 and filtered for sterilization to obtain lactoferrin-modified norcantharidin liposome injection (NCTD-Lips-Lf).

[0067] In a specific embodiment, step (4) specifically comprises: EDC and NHS are mixed according to EDC, NHS and DSPE-PEG 2000 -COOH was added to NCTD-Lips in a molar ratio of 9:9:1 to 12:12:1, and stirred at room temperature for 15 min. 2000Lactoferrin was added in a ratio of 1:35 to -COOH molar ratio, incubated at 35°C to 40°C for 2h to 4h, and then a dialysis bag with a molecular weight cutoff of 10KDa was used. The dialysis medium was a sodium sulfate aqueous solution with a pH of 7.4 to remove free drugs, EDC, and NHS, and the mixture was sterilized by filtration using a filter membrane with a diameter of 0.22μm and stored at 4°C.

[0068] The present invention also discloses the use of the above-mentioned norcantharidin liposome injection or the norcantharidin liposome injection prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0069] In one embodiment, the tumor comprises liver cancer.

[0070] The following are specific embodiments

[0071] Example 1-Example 10

[0072] The present invention proposes 10 groups of embodiments of preparing sodium norcantharidin liposome injection using the preparation method of the present invention. The specific mass ratios of the raw material components of Examples 1-10 are shown in Table 1, the specific process parameters are shown in Table 2, and the preparation process is as follows:

[0073] (1) dissolving norcantharidin in water at 60-100° C., adding alkali to adjust the pH to 9-11, heating at 60-80° C. for 20 min-40 min, and then adding acid to adjust the pH to 3-4 to obtain pretreated norcantharidin.

[0074] (2) Weigh the prescribed amount of phospholipids and DSPE-mPEG 2000 , cholesterol and DSPE-mPEG 2000 -COOH was dissolved in chloroform and evaporated under reduced pressure at 40°C to obtain a lipid film. A calcium acetate aqueous solution was added to hydrate the film at 55-65°C for 15-60 min. The obtained large unilamellar liposomes were filtered through 0.45 μm and 0.22 μm filter membranes three times respectively. Finally, the liposome particle size was controlled, and the whole liposomes were dialyzed through a sodium sulfate aqueous solution with a certain pH to remove calcium ions in the external aqueous phase, and the external liquid was switched to a dialysis medium to form a pH gradient inside and outside the liposomes to obtain blank liposomes.

[0075] (3) Blank liposomes and pretreated norcantharidin were mixed for drug loading to obtain NCTD-Lips.

[0076] (4) EDC and NHS were divided into EDC, NHS and DSPE-PEG 2000-COOH was added to NCTD-Lips in a molar ratio of 9:9:1 to 12:12:1, and after stirring at room temperature for 15 minutes, lactoferrin was added and incubated at 37°C for 3 hours. Then, a dialysis bag with a molecular weight cutoff of 10 KDa and a dialysis medium of sodium sulfate aqueous solution at pH 7.4 were used to remove free drugs, EDC, and NHS, and the filter membrane with a diameter of 0.22 μm was used for filtration and sterilization, and the mixture was stored at 4°C to obtain NCTD-Lips-Lf.

[0077] Table 1: Raw material components by weight in Examples 1-10

[0078]

[0079]

[0080] Table 2 Preparation process parameters of Examples 1-10

[0081]

[0082] The structural layer test was performed on the NCTD-Lips-Lf or NCTD-Lips prepared in Example 1 to Example 10. The results are shown in Table 3.

[0083] Table 3 Preparation parameters of Examples 1-10

[0084]

[0085] The NCTD-Lips-Lf prepared in Example 10 and the NCTD-Lips prepared in Example 9 were characterized as follows:

[0086] The particle size and potential information of NCTD-Lips-Lf and NCTD-Lips are shown in Table 4. The particle size of NCTD-Lips liposomes is about 102nm, but the particle size of NCTD-Lips-Lf group is slightly larger, which may be related to the connection of lactoferrin. The potential of the two liposomes is -16mV to -19mV, and the pH of the two liposomes is about 7.4. The encapsulation efficiency of NCTD-Lips-Lf (Table 3) is as high as 99.3%.

[0087] like Figure 3 As shown, the liposome injection solution is a milky white homogeneous solution with opalescence. The transmission electron microscopy results are as follows Figure 4 The results show that the NCTD-Lips-Lf and NCTD-Lips liposomes are of uniform size and present a complete spherical state. Figure 5 The results of Malvern particle size determination are shown.

[0088] X-ray diffraction results ( Figure 6 ) showed that pure NCTD exhibited a series of strong crystal characteristic diffraction peaks (2θ=15.7°and 33.3°), indicating that pure NCTD had good crystallinity, while in NCTD-Lips-Lf and NCTD-Lips samples, the drug characteristic peaks disappeared, indicating that norcantharidin acid was successfully encapsulated inside the liposomes.

[0089] Table 4 Parameters of preparations prepared in Example 9 and Example 10

[0090]

[0091] Test Example 1: The stability of NCTD-Lips-Lf prepared in Example 10 and NCTD-Lips prepared in Example 9 were tested.

[0092] Experimental method: NCTD-Lips-Lf and NCTD-Lips were added to PBS buffer containing 10% fresh plasma and shaken in a 37°C water bath. 100 μL samples were taken at 0.5, 1, 2, 4, 6, 8, 12 and 24 h, and the particle size was measured using a Malvern laser particle size analyzer. At the same time, 500 μL samples were taken and the NCTD content in NCTD-Lips-Lf and NCTD-Lips was determined by HPLC after treatment. At the same time, appropriate amounts of NCTD-Lips-Lf and NCTD-Lips were taken and stored at 4°C in the dark, and samples were taken on day 0, 7, 14, 21, 30, 60 and 90 to determine the liposome particle size and the content of norcantharidin.

[0093] The particle size and encapsulation efficiency of NCTD-Lips-Lf prepared in Example 10 and NCTD-Lips prepared in Example 9 in PBS buffer containing 10% fresh plasma are shown in the following table. Figure 7 As shown in the figure, the particle size of the two liposomes fluctuated within 24 hours, but the overall change in the average particle size did not exceed 10%, indicating that the liposomes had good physical stability in 10% plasma medium. In addition, during the incubation process, the content of norcantharidin in the two liposomes was also maintained at more than 95%, indicating that the two liposomes also had good chemical stability in plasma, which laid the foundation for subsequent in vivo experiments. After NCTD-Lips-Lf and NCTD-Lips were stored at 4°C for three months, the particle size remained almost unchanged, and the content of norcantharidin still remained above 95%, indicating that both liposomes had good stability and could be stored for a long time at low temperature and in the dark.

[0094] Test Example 2: The hemolytic properties of NCTD-Lips-Lf prepared in Example 10 and NCTD-Lips prepared in Example 9 were tested.

[0095] Experimental method: Take fresh rat blood and prepare 2% red blood cell suspension (v / v) with physiological saline. Add each test solution according to the proportion in Table 5, and incubate the sample in a 37℃ water bath for 3h, centrifuge at 3000rpm for 10min, and observe whether there is hemolysis in each tube. At the same time, use an enzyme marker to measure the absorbance of the sample at 545nm, and calculate the hemolysis rate according to the following formula. Finally, observe the red blood cell status of each group of samples under a microscope.

[0096]

[0097] Table 5 Volume of each component in hemolysis experiment

[0098]

[0099] Table 6 Hemolysis rate results of each group.

[0100]

[0101] The test results are as follows Figure 8 As shown in Table 6, the color of the self-made NCTD and NCTD-Lips-Lf and NCTD-Lips hemolysis test samples was comparable to that of the negative control samples of the corresponding concentrations, indicating that hemolysis did not occur. The UV absorbance results are shown in Table 6. The hemolysis rate of each preparation was calculated to be no higher than 2%. In addition, the cell morphology of each preparation was intact under the microscope ( Figure 8 ), while the cells in the positive control group showed obvious swelling and rupture of red blood cells, further proving that each preparation would not produce hemolytic reaction on red blood cells. This provides a basis for subsequent pharmacokinetic and pharmacodynamic studies.

[0102] Test Example 3: Testing the tissue distribution of NCTD-Lips-Lf prepared in Example 10 and NCTD-Lips prepared in Example 9 after administration to mice bearing liver cancer H22 tumor strain.

[0103] Experimental methods:

[0104] Preparation of fluorescent preparation: First, weigh the prescribed amount of DSPC, cholesterol, DSPE-mPEG 2000 , DSPE-mPEG 2000 -COOH and 1 mg of DiR were placed in a round-bottom flask, and DiR-loaded liposomes were prepared by thin film hydration method, and the particle size was controlled to about 100 nm by probe ultrasound, and then covalently coupled to lactoferrin to obtain DiR-Lips-Lf. At the same time, DiR-Lips without lactoferrin modification was prepared by the same method.

[0105] Establish a mouse model bearing liver cancer H22 tumor strain and wait until the tumor volume increases to 100mm 3As mentioned above, the prepared DiR-Lips, DiR-Lips-Lf and DiR-sol (DiR as a fluorescent agent) were injected through the tail vein at a concentration of 2 mg / kg. At 2h, 6h, 12h and 24h, the mice were immediately killed by dislocating the neck, and the heart, liver, spleen, lungs, kidneys and tumors were removed. They were rinsed with saline and the surface saline was absorbed with filter paper. The mice were placed in a fluorescent in vivo imaging system for scanning and imaging, and the scanning imaging results were semi-quantitatively analyzed. The acquisition parameters are as follows: excitation wavelength 720nm, emission wavelength 790nm, temperature 36.5℃, exposure time 30s. The test results are as follows: Fig. 9 As shown in the figure, after DiR-Lips, DiR-Lips-Lf and DiR-sol were injected into the tail vein of mice, DiR-Sol was rapidly distributed in various organs. Compared with the two liposome preparations loaded with DiR fluorescent dyes, DiR-Sol was quickly metabolized and cleared, and had no tumor targeting, while the DiR-Lips and DiR-Lips-Lf groups still had strong fluorescence 24 hours after administration, indicating that the two liposome preparations were slowly metabolized and cleared in vivo. This is mainly due to the hydrophilic PEG modified on the surface of the liposomes, which reduces the effect of plasma protein opsonin on the drug, reduces the recognition and phagocytosis of macrophages in the reticuloendothelial system, and thus prolongs the circulation time of the drug in vivo. In addition, DiR-Lips-Lf not only has a longer circulation time in vivo, but also has a relatively stronger fluorescence than the DiR-Lips group, indicating that it has a more obvious tumor targeting, which is conducive to improving the treatment effect and reducing damage to other organs.

[0106] Test Example 4: Testing the in vivo pharmacokinetics of NCTD-Lips-Lf prepared in Example 10 and NCTD-Lips prepared in Example 9 after administration to SD rats.

[0107] Dosage: The clinical dosage of sodium norcantharinate injection is 10-30 mg once a day. According to the "Pharmacological Experimental Design and Statistical Analysis", the equivalent dose for rats is 0.9-2.7 mg / kg. Referring to the dose selection item of the "Technical Guidelines for Nonclinical Pharmacokinetic Studies of Chemical Drugs", the dosage for the medium-dose group of rats is 2.7 mg / kg, the dosage for the high-dose group is 5.4 mg / kg, and the dosage for the low-dose group is 1.35 mg / kg. Finally, 2.7 mg / kg was selected as the dosage for rats in this pharmacokinetic study.

[0108] Sample collection: SD rats were randomly divided into three groups, 5 rats in each group, and NCTD-Lips, NCTD-Lips-Lf and commercially available NCTD-sol (2.7 mg / kg) were injected into the tail vein of the rats. 0.5 mL of blood was collected from the eye sockets at specific time points (0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 and 48 h), centrifuged at 8000 rpm for 10 min, and the upper plasma was taken to detect the concentration of norcantharidin acid in the plasma using liquid chromatography-mass spectrometry.

[0109] Test results such as Fig.10 As shown in Table 7, Fig.10 The plasma concentration-time curves of NCTD, NCTD-Lips-Lf and NCTD-Lips (n=5)

[0110] Table 7 In vivo pharmacokinetic parameters after intravenous injection of NCTD, NCTD-Lips-Lf and NCTD-Lips. Data are presented as mean ± SD (n = 5).

[0111]

[0112] The pharmacokinetic curves were fitted using the statistical moment model using DAS2.0 statistical software to obtain the pharmacokinetic parameters of each group. The experimental results are shown in Table 7. The area under the drug-time curve (AUC) of the NCTD-Lips-Lf and NCTD-Lips groups 0-t ) were 120837.3±18165.1ug / L*h and 207794.8±23524.3ug / L*h, respectively, which was the AUC of NCTD-Sol (28168.5±22791.1ug / L*h) group. 0-t The mean elimination half-life (t 1 / 2 ) were 11.911±0.382h and 27.596±1.332h, which were significantly higher than the t of NCTD-Sol. 1 / 2(2.303±0.121h). At the same time, the plasma clearance (CL) of the NCTD-Lips-Lf and NCTD-Lips groups were 0.023±0.005L / h / kg and 0.011±0.002L / h / kg, respectively, which were significantly lower than that of the NCTD-Sol group (0.106±0.024). In summary, liposomes improve the in vivo pharmacokinetic characteristics of drugs by prolonging the retention time of NCTD in the body, reducing the clearance rate and improving the bioavailability. Using liposomes as carriers to deliver NCTD, on the one hand, can protect the drug by encapsulating the drug in the phospholipid bilayer, preventing the degradation of the drug by plasma endogenous substances and improving stability; on the other hand, modifying the hydrophilic PEG on the surface of the liposome can reduce the effect of plasma protein opsonins on the drug, reduce the recognition and phagocytosis of macrophages in the reticuloendothelial system, thereby prolonging the circulation time of the drug in the body, which is beneficial to improve the therapeutic effect.

[0113] Compared with the NCTD-Lips group, the AUC 0-t The half-life is relatively short, which may be due to the fact that the lactoferrin modified on the surface of liposomes is easily recognized by endogenous substances in the plasma, which destroys the liposome structure and leads to the degradation of NCTD. In addition, the lactoferrin on the surface of liposomes has a high affinity with the ASGPR receptor highly expressed on the surface of liver cancer cells, which makes the lactoferrin-modified liposomes specifically target tumor cells, thereby reducing the distribution of liposomes in the blood.

[0114] Test Example 5: Comparative test of the pharmacodynamics and safety of NCTD-Lips-Lf prepared in Example 10, NCTD-Lips prepared in Example 9 and commercially available sodium norcantharinate injection after administration to mice bearing liver cancer H22 tumor strain.

[0115] Experimental plan: Tumor-bearing mice were divided into 4 groups, namely NCTD-Lips-Lf group, NCTD-Lips group, commercially available sodium norcantharidinate injection (Na2DCA) and normal saline, with 6 mice in each group. Each group was injected with drugs through the tail vein, and the dosage was sodium norcantharidinate (2.7 mg / kg). The dosing schedule was as follows Fig.11 The body weight and tumor volume of mice were monitored daily as indicated.

[0116] During the experiment, the longest diameter (a) and shortest diameter (b) of the tumor were measured with a vernier caliper, and the tumor volume (TV) was calculated. On the 14th day of the experiment, the experimental mice were killed, the tumors were dissected and separated, weighed, and the tumor inhibition rate (TIR) ​​was calculated.

[0117] Finally, the mouse serum was collected for biochemical analysis to examine liver function, kidney function and myocardial enzymes. At the same time, the tumor-bearing mice were killed by cervical dislocation, and the main organs (heart, liver, spleen, lung, kidney) and tumors were removed and fixed in 4% paraformaldehyde. Then, after the tissues were processed and stained with hematoxylin-eosin, the pathological changes of each tissue were observed under a microscope.

[0118] Test results such as Fig.12 As shown in the results, compared with the saline group, the NCTD-Lips preparation group had a significant inhibitory effect on tumor growth (P<0.01), with a tumor inhibition rate of 59.13%; the NCTD-Lips-Lf group and the NCTD-Lips group had a very significant inhibitory effect on tumors (P<0.001), with tumor inhibition rates of 85.29% and 83.57%, respectively. The anti-tumor activity of the NCTD-Lips-Lf group was slightly better than that of the NCTD-Lips group, which may be due to the modification of lactoferrin, which makes norcantharidin acid more distributed in tumor cells in the body, so the anti-tumor effect is better.

[0119] Fig.13 Serum biochemical analysis, including liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP)), renal function (urea nitrogen (BUN), creatinine (CRE)), and cardiac enzymes (creatine kinase (CK), lactate dehydrogenase (LDH, LDH-1)) were shown. Compared with the baseline parameters of healthy mice, all the parameters measured in each group were within the normal range. There was no statistical difference in the liver function and renal function parameters between the treatment groups and the control group. Therefore, the damage to renal and liver function in each treatment group was negligible. Compared with the blank control group, the creatine kinase (CK) concentration was significantly increased in the NCTD-Lips group (P<0.001) and the positive control group (P<0.01). This suggests that NCTD-Lips may induce a certain degree of cardiotoxicity. In contrast, lactoferrin-modified liposomes can significantly enhance the tumor targeting of NCTD and reduce the accumulation of NCTD in cardiac tissue, thereby significantly reducing the associated cardiotoxicity.

[0120] Fig.14 The following are hematoxylin-eosin (HE) staining images of all organs and tumors. Some myocardial fiber ruptures were observed in the myocardial tissue sections of the NCTD-Lips group and the positive control group. No damage was observed in the organs of the NCTD-Lips-Lf group and the blank control group, which was consistent with the results of serum biochemical analysis. In addition, the tumor tissue structure of mice treated with normal saline was intact, the tumor cells were dense, the cell nuclei were regular in morphology, and the entire tissue section had a bright appearance. The tumor tissues of the NCTD-Lips group and the NCTD-Lips-Lf group were damaged to varying degrees, with the most obvious damage in the NCTD-Lips-Lf group, which was manifested by shrinkage and loose arrangement of tumor cells, and atrophy and disintegration of cell nuclei.

[0121] Test Example 6: Comparative study on the vascular irritation of NCTD-Lips-Lf prepared in Test Example 10 and commercially available sodium norcantharidin injection after administration to mice.

[0122] Experimental method: BALB / C mice were randomly divided into 4 groups, and NCA-Lips-Lf, commercially available Na2DCA injection and saline were injected into the tail vein of the mice at the same dose (3 mg / kg in terms of norcantharidin), and the remaining group was not treated (untreated). The drug was administered once a day for 7 consecutive days, and the status of the tail vein of the mice was observed every day to evaluate the irritation of each preparation to the tail vein of the mice.

[0123] The test results are as follows Fig.15 As shown in the figure, compared with the untreated group, the tails of mice in the NCTD-Lips-Lf group and the saline group were only slightly red (p<0.05). The tail veins of mice in the sodium norcantharidin injection (Na2DCA) group showed severe redness and swelling, and the tail vein blood vessels turned dark black (p<0.001), indicating strong vascular irritation. The above results show that the encapsulation of drugs by norcantharidin acid liposomes can reduce their irritation to blood vessels to a certain extent, and the appropriate pH value of the external aqueous phase of the liposome preparation (pH about 7.4) can effectively avoid the irritation to the blood vessel wall caused by the excessively high pH value of the commercially available sodium norcantharidin injection (pH 9-10).

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A norcantharidin liposome injection, characterized in that: The composition includes the following components in parts by weight: 5 to 15 parts of norcantharidin, 50 to 200 parts of phospholipids, 30 to 40 parts of DSPE-mPEG 2000 , 25 to 50 parts of cholesterol and 79.1 to 118.6 parts of calcium acetate.

2. The norcantharidin liposome injection according to claim 1, characterized in that: The mass ratio of the phospholipid to the cholesterol is 2:1 to 4:1; The phospholipid and the DSPE-mPEG 2000 The molar ratio is 12:1 to 10:1; The phospholipids include one or more of S100, E80, PC-98T, DSPC and SM.

3. The norcantharidin liposome injection according to claim 1, characterized in that: The following components are also included: 2.5 to 5.3 parts of DSPE-mPEG 2000 -COOH and 1.8 to 3.2 parts of lactoferrin.

4. The norcantharidin liposome injection according to claim 3, characterized in that: The DSPE-mPEG 2000 With the DSPE-mPEG 2000 The molar ratio of -COOH is 7:1 to 12:

1. The lactoferrin and the DSPE-PEG 2000 The molar ratio of -COOH is 1:30 to 1:

50.

5. The norcantharidin liposome injection according to claim 3, characterized in that: The medicine-lipid ratio (w / w) of the norcantharidin liposome injection is 1:10-1:20, the average particle size of the norcantharidin liposome is 100nm-200nm, the 90% particle size cumulative value is ≤220nm, the content is 95%-105% of the labeled amount, the encapsulation rate is 85%-99.9%, and the lactoferrin connection rate is 40%-65%.

6. A method for preparing the norcantharidin liposome injection according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) dissolving norcantharidin in water at 60° C. to 100° C., adding alkali to adjust the pH to 9 to 11, heating at 60° C. to 80° C. for 20 min to 40 min, and then adding acid to adjust the pH to 3 to 4 to obtain pretreated norcantharidin; (2) Phospholipids and DSPE-mPEG 2000 and cholesterol are dissolved in an organic solvent, and the organic reagent is removed by evaporation under reduced pressure to obtain a lipid film, and then calcium acetate is added for hydration and particle size treatment to obtain large unilamellar liposomes, and then the outer aqueous phase of the large unilamellar liposomes is replaced with a sodium sulfate aqueous solution with a pH of 3 to 7 to form a pH gradient inside and outside the liposome membrane to obtain blank liposomes; (3) Mixing the blank liposomes and the pretreated norcantharidin for drug loading to obtain the norcantharidin liposome injection.

7. The preparation method according to claim 6, characterized in that: In step (2), the concentration of calcium acetate is 100 mM to 150 mM; The organic solvent includes one or more of chloroform, methanol and dichloromethane; The hydration temperature is 55°C to 65°C; the hydration time is 15min to 60min; The granulation specifically comprises: sequentially treating with a filter membrane with a diameter of 0.45 μm and a filter membrane with a diameter of 0.22 μm, and then using an extrusion method or a probe ultrasonic method to obtain a large single-layer liposome with a particle size of 100 nm to 200 nm; In step (3), the drug loading temperature is 55°C to 65°C, and the drug loading time is 20min to 60min.

8. The preparation method according to claim 6, characterized in that: The preparation method further comprises the following steps: The norcantharidin liposomes obtained in step (3) are mixed with DSPE-mPEG 2000 After mixing -COOH, NHS and EDC, lactoferrin was added for incubation to allow the amino groups on lactoferrin to react with DSPE-mPEG. 2000 The carboxyl group on -COOH is covalently coupled, and then the pH of the external aqueous phase is adjusted to 7.4 and filtered for sterilization to obtain a lactoferrin-modified norcantharidin liposome injection.

9. The preparation method according to claim 8, characterized in that: EDC, NHS, and DSPE-PEG 2000 The molar ratio of -COOH is 9:9:1 to 12:12:1; The incubation temperature is 35°C to 40°C; the incubation time is 2h to 4h; The diameter of the filter membrane used in the filtration sterilization is 0.22 μm.

10. Use of the norcantharidin liposome injection according to any one of claims 1 to 5, or the norcantharidin liposome injection prepared by the preparation method according to any one of claims 6 to 9 in the preparation of antitumor drugs.

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