CN104840968A - Docetaxel nano-micelle preparation carried by methyl polyethylene glycol2000-b-poly D, L-lactic acid1000-1500block copolymer - Google Patents

Abstract

The invention relates to a polymer nanomicelle preparation loaded with insoluble antitumor drug docetaxel, a preparation method and application thereof. The micellar preparation of the present invention is methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000-1500 block copolymer loaded docetaxel micelles, the particle diameter range is 15nm～25nm, the dispersion coefficient of the particle is PI is between 0.09 and 0.15. The micellar preparation of the present invention has the following advantages: the particle size of the micelles is small, the distribution range is concentrated, the encapsulation efficiency is high, the drug loading capacity is high, it is stable after freeze-drying, and the reconstitution stability is good, which can obviously improve the concentration of docetaxel. Water-soluble, significantly improves the therapeutic effect of docetaxel on metastatic breast cancer and pancreatic cancer, and has low toxic and side effects. The micellar formulation of the present invention can be specially prepared as a lyophilized formulation for intravenous injection.

Description

Methyl polyethylene glycol 2000-b-poly D,L-lactic acid 1000-1500 block copolymer loaded docetaxel nanomicelle preparation

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000-1500 micelle preparation loaded with an insoluble antitumor drug docetaxel, a preparation method and an application thereof.

Background technique

Docetaxel (docetaxel, trade name etc.) are a new generation of taxane antineoplastic drugs, which are obtained by semi-synthesis from the inactive compounds extracted from the needles of European berry yew (Taxus baccata). By binding to tubulin, it stops tumor cells from mitotic division and dies, and effectively inhibits the replication of tumor cells. Clinical studies have shown that docetaxel can be used alone or in combination with other antineoplastic drugs to treat non-small cell lung cancer, breast cancer, ovarian cancer, gastric cancer and other cancers. Due to the poor water solubility of docetaxel, most of the existing injections use Tween-80 as solvent, and at the same time, it is equipped with a solvent containing 13% ethanol, which is diluted before use, and the tumor targeting is not high.

Tween-80 is hemolytic and viscous, so it is easy to cause severe allergic reactions after injection of commercially available preparations, and the irritation reaction is large. The incidence of moderate to severe allergies is as high as 25.9%. It is used in doses of 75mg/m ² and 100mg/m ² The incidences of moderate and above allergies were 31% and 41.3%, respectively, and the injection site reaction rate was as high as 13.3%. Therefore, anti-allergic treatment is required when docetaxel is used clinically. In addition, the clinical use of docetaxel is also quite cumbersome. During the infusion process, it is necessary to closely observe whether there is any leakage at the infusion site. If there is leakage, the injection site must be replaced immediately and partially sealed. At the same time, routine ECG monitoring is required during the medication period, closely observe changes in breathing, heart rate, and blood pressure, be highly alert to the occurrence of allergic reactions, and be prepared for medical rescue of severe allergic reactions. At present, reducing the toxic and side effects of docetaxel preparations and improving the therapeutic effect is a technical difficulty of widespread concern, and it is also a worldwide technical difficulty.

The study of polymer micelles is one of the important fields in the study of pharmaceutical nanosystems, which has developed rapidly in recent years. Since polymer micelles have the advantages of low critical micelle concentration, large solubilization space, and good thermodynamic stability, and the different properties of polymer hydrophobic blocks, various methods such as chemical, physical, and electrostatic interactions can be used. Encapsulating drugs makes the polymer micelles have broad application prospects in the field of drug carriers. Polylactic acid (PLA) is a biodegradable material, and its final metabolites are carbon dioxide and water. The intermediate metabolite lactic acid is also a normal metabolite in the body and will not accumulate in the body. Polyethylene glycol (PEG) is a commonly used hydrophilic block. As a polyether polymer, PEG has good hydrophilicity and biocompatibility. However, there is no literature in the prior art teaching that a high molecular block polymer synthesized by combining hydrophobic PLA and PEG, having both a hydrophilic segment and a hydrophobic segment, can be used as a Micellar carrier for docetaxel.

The parameters for evaluating the polymer micellar system include particle size, drug loading and stability. The micelles containing docetaxel have been reported in the world, and the drug loading is generally about 5%, and almost no more than 10%, and there are few studies on the stability of freeze-dried preparations, which greatly limits the ability of docetaxel gels. The real application of beam in clinical aspect.

Contents of the invention

An object of the present invention is to overcome the defects of the prior art and provide a methyl polyethylene glycol ₂₀₀₀ -b-poly D,L-lactic acid _1000-1500 (mPEG _2000- b-PDLLA _1000～1500 ) micellar preparation. The micelle preparation comprises antineoplastic drug docetaxel, amphiphilic block copolymer methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000-1500 %, and optional pharmaceutically acceptable jelly Dry protectant.

In the present invention, the micellar carrier material selects mPEG ₂₀₀₀ -b-PDLLA _1000-1500 as the carrier material. The present invention also provides a preferred synthesis method of mPEG ₂₀₀₀ -b-PDLLA _1000-1500 , the method is: after removing water from mPEG ₂₀₀₀ under reduced pressure, add 0.5% (w/w) stannous octoate, and stir at 110°C 0.5 hours, then add D,L-lactide, react for 5 hours under _N2 protection and heating at 130°C, after standing at room temperature, dissolve with dichloromethane, then drop into -20°C and freeze for 2 hours In the above diethyl ether, filter the precipitated product, dry it under reduced pressure at room temperature, and repeat the post-treatment work (dissolve in dichloromethane and drop into frozen diethyl ether) twice to obtain the product. The molecular weight of the PDLLA block is 1000-1500 as measured by ¹ H-NMR. The carrier material is preferably mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ block polymer.

In the present invention, when a lyoprotectant is added, the lyoprotectant is PEG2000, Poloxamer 188 (Poloxamer188), lactose, glucose, sorbitol, mannitol, mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ , human blood A mixture of any one or more of albumin (HSS), PVP K30, preferably a mixture of one or more of glucose, mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ , and HAS. The added amount of the lyoprotectant is 1-10% by mass based on the total amount of the lyophilized preparation, preferably 5% by mass.

In the present invention, the mass ratio of docetaxel to mPEG ₂₀₀₀ -b-PDLLA _1000-1500 is 1:3-1:9, preferably 1:5.

The drug loading of the nanomicelle preparation of the present invention is between 10% and 25%, preferably 15% to 18%; the diameter of the nanoparticles after reconstitution is between 15nm and 25nm, and the dispersion coefficient P.I. of the particles is between 0.09 and 0.15 between; the encapsulation efficiency of docetaxel in the micelles reaches at least 85%. The nano-micelle preparation of the present invention can be in a freeze-dried form, that is, a freeze-dried nano-micelle preparation, which can be used for intravenous injection.

A further object of the present invention is to provide a preparation method of the docetaxel micellar preparation.

The preparation method of the mPEG ₂₀₀₀ -b-PDLLA _1000-1500 micelle freeze-dried preparation loaded with docetaxel of the present invention comprises the steps of:

(1) Preparation of docetaxel-loaded micellar aqueous solution by film hydration method;

Dissolve docetaxel and mPEG ₂₀₀₀ -b-PDLLA _1000-1500 in an organic solvent, remove the organic solvent to obtain a gel-like drug-containing lipid film, then add an aqueous solution to dissolve and disperse the drug-containing lipid film to obtain a micellar solution;

(2) The micelle solution (optionally adding lyophilized excipients) is sterilized, filtered, and lyophilized to prepare a lyophilized preparation of drug-loaded polymer micelles.

In a specific scheme, the preparation method of docetaxel nano-micelle preparation of the present invention comprises the steps:

(1) Docetaxel and methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000-1500 are dissolved in an organic solvent;

(2) remove the organic solvent to obtain a polymer lipid film containing docetaxel;

(3) Add water for injection and hydrate at 25°C to 60°C;

(4) Vortex or ultrasonic to obtain a nanomicelle solution;

(5) Optionally add a lyoprotectant, filter and sterilize with a 0.22 μm sterile microporous membrane, freeze in a refrigerator at -50°C to -70°C for 12 hours, and then freeze-dry to obtain docetaxel-loaded Methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000~1500 nanometer micelles preparation.

In a more specific embodiment, in step (2), the organic solvent is removed by reducing pressure and/or removing the organic solvent under vacuum; Hydrate for 1-2 hours; vortex or sonicate for 1-5 minutes in step (4).

In the present invention, the organic solvent refers to an organic solvent that can completely dissolve mPEG ₂₀₀₀ -b-PDLLA _1000-1500 and docetaxel, including acetonitrile, methanol, ethanol and chloroform, etc. The above-mentioned solvents can be used alone or in combination Use; the organic solvent is preferably acetonitrile; the ratio of the added volume (ml) of the organic solvent to the total amount (mg) of the drug and excipients is 1:5-1:60, preferably 1:60.

In the present invention, the dispersion solution for dissolving the drug film (ie, drug-containing lipid film) is selected from water for injection.

In the present invention, the micellar solution can be directly freeze-dried, or freeze-dried after adding a freeze-drying protective agent.

The freeze-dried preparation can use water for injection, 5% glucose injection or 0.9% sodium chloride injection as the dispersion medium for clinical use.

In the present invention, the size range of micelles before freeze-drying and after reconstitution is 15-25 nm, and the dispersion coefficient P.I is between 0.09-0.15.

The reagents and raw materials used in the present invention are all commercially available, or obtained by the preparation method recorded in the description (if the preparation method is described in the description); for the devices, conditions (temperature, time, etc.) not specifically described in the present invention, Substances, dosages, methods, etc., can all be known in the art or determined by those skilled in the art according to conventional techniques.

Another object of the present invention is to provide the application of the freeze-dried preparation in the preparation of medicines for treating metastatic breast cancer or pancreatic cancer. The present invention takes mouse metastatic breast cancer 4T1 and Capan-2 pancreatic cancer as tumor research models, and shows that docetaxel micelles can more effectively inhibit the metastasis of cancer cells than the existing commercial preparations of docetaxel, and improve the therapeutic effect. effect, while reducing systemic toxicity.

Due to the deep anatomical location and hidden symptoms of pancreatic cancer, it is not easy to detect early; even if small pancreatic cancer can be detected by imaging, a considerable part has lymphatic metastasis and nerve invasion, and it is easy to relapse and metastasis after operation. Neither are sensitive. The traditional first-line chemotherapy drug is only gemcitabine; and taxanes, especially docetaxel, are the most widely used chemotherapy drugs in the treatment of secondary metastases and refractory tumors. The present inventors have found that using the docetaxel nanomicelle preparation of the present invention for the treatment of pancreatic cancer or metastatic pancreatic cancer can achieve unexpected therapeutic effects, which provides a basis for the application of taxanes in the treatment of pancreatic cancer. important reference.

The mPEG ₂₀₀₀ -b-PDLLA _1000-1500 micelle freeze-dried preparation loaded with docetaxel of the present invention has the following advantages:

1. The selected mPEG ₂₀₀₀ -b-PDLLA _1000～1500 block polymer material is non-toxic, non-immunogenic, biodegradable in vivo, and has good biocompatibility; it is suitable for the entrapment and solubilization of docetaxel It is realized by utilizing its self-assembly in aqueous solution to form micelles, without the use of Tween-80 and other solubilizers, eliminating the hidden dangers of drug safety caused by traditional solvents, and greatly improving the safety of docetaxel;

2. The prepared mPEG ₂₀₀₀ -b-PDLLA _1000～1500 micelle freeze-dried preparation loaded with docetaxel can significantly improve the water solubility of docetaxel, and has high encapsulation efficiency (>90%) and high drug loading capacity (10%~25%), small particle size (15~25nm), narrow dispersion coefficient (0.09<PI<0.15), concentrated distribution range, and improved bioavailability; the freeze-drying process significantly improved the loading of polyene The stability of paclitaxel micelles facilitates the storage and transportation of preparations;

3. It is convenient for clinical use. After adding water for injection, normal saline or 5% glucose injection, etc., the freeze-dried preparation can be quickly dissolved and dispersed into a transparent micellar solution with low viscosity and good fluidity. The change in particle size within one hour is less than 1%, which greatly improves the safety of medication and fully meets the clinical medication needs;

4. The preparation method of the present invention is simple and easy, with mild and controllable conditions and good repeatability; by controlling the dosage and the volume of the dispersion solution, a series of drug-loaded micelles with different concentrations can be obtained, which is easy to control the production process and industrialized production implementation of

5. It is confirmed by in vitro cytotoxicity experiments and animal in vivo experiments that the docetaxel micellar preparation of the present invention can significantly improve the therapeutic effect against pancreatic cancer and metastatic breast cancer, reduce the systemic toxicity of docetaxel, and has good clinical efficacy. Value.

Description of drawings

Fig. 1 is the ¹ H-NMR spectrum of the block structure of mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ block copolymer in Preparation Example 2 of the present invention.

Fig. 2 is the permeation gel chromatography (GPC) spectrum of mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ block copolymer in Preparation Example 2 of the present invention.

Figure 3 shows the dynamic light scattering (DLS) particle size distribution diagram of the docetaxel micelles of the present invention measured by a laser particle size analyzer.

Fig. 4 is a transmission electron micrograph (TEM) of the docetaxel-loaded micelles of the present invention.

Fig. 5 is a graph showing the change in particle size of the solution within 6 hours after reconstitution of the drug-loaded micelles after adding various lyoprotectants of the present invention.

Fig. 6 is an in vitro release curve of drug-loaded micelles in PBS solution.

Fig. 7 is a diagram showing the results of MTT experiments of commercially available docetaxel, docetaxel micelles and blank micelles.

Fig. 8 is a diagram showing the effect of docetaxel micelles of the present invention on the body weight change of Balb/c mice bearing 4T1 tumors.

Fig. 9 is a comparison chart of HE staining of the lungs of Balb/c mice bearing 4T1 tumors with commercially available docetaxel and docetaxel micelles of the present invention.

Fig. 10 is a comparison chart (a) and a body weight change curve (b) of the therapeutic effects of commercially available docetaxel and docetaxel micelles of the present invention on pancreatic cancer.

Detailed ways

The present invention is further illustrated by way of examples below, but it does not mean that the present invention is limited within the scope of the described examples.

Preparation Example 1 Synthesis of mPEG ₂₀₀₀ -b-PDLLA ₁₀₀₀

After removing water from 2 g of mPEG ₂₀₀₀ under reduced pressure, add 0.5% (w/w) stannous octoate, stir at 110 °C for 0.5 h, then add 1.1 g of D,L-lactide, under _N2 protection and 130 °C Under heating conditions, react for 5 hours, after standing at room temperature, dissolve with dichloromethane, then drop into diethyl ether frozen at -20°C for 2 hours, filter the precipitated product, dry under reduced pressure at room temperature, and post-process (use Dichloromethane was dissolved and dropped into ice-cold ether) repeated twice to obtain 2 g of the product. The molecular weight of the PDLLA block was 1000 as measured by ¹ H-NMR.

Preparation example 2 mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ synthesis

After removing water from 2 g of mPEG ₂₀₀₀ under reduced pressure, add 0.5% (w/w) stannous octoate, stir at 110 °C for 0.5 h, then add 1.4 g of D,L-lactide, under _N2 protection and 130 °C Under heating conditions, react for 5 hours, after standing at room temperature, dissolve with dichloromethane, then drop into diethyl ether frozen at -20°C for 2 hours, filter the precipitated product, dry under reduced pressure at room temperature, and post-process (use Dichloromethane was dissolved and dropped into ice-cold ether) repeated twice to obtain 2.1 g of the product. The molecular weight of the PDLLA block was determined to be 1300 by ¹ H-NMR (Figure 1), and its polydispersity coefficient was 1.20 by GPC analysis (Figure 2).

Preparation Example 3 Synthesis of mPEG ₂₀₀₀ -b-PDLLA ₁₅₀₀

After removing water from 2 g of mPEG ₂₀₀₀ under reduced pressure, add 0.5% (w/w) stannous octoate, stir at 110 °C for 0.5 h, then add 1.7 g of D,L-lactide, under _N2 protection and 130 °C Under heating conditions, react for 5 hours, after standing at room temperature, dissolve with dichloromethane, then drop into diethyl ether frozen at -20°C for 2 hours, filter the precipitated product, dry under reduced pressure at room temperature, and post-process (use Dichloromethane was dissolved and dropped into ice-cold ether) repeated twice to obtain 2.1 g of the product. The molecular weight of the PDLLA block was 1500 as measured by ¹ H-NMR.

Embodiment loads the preparation of docetaxel polymer micelle preparation

Dissolve docetaxel and mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ in 5 mL of acetonitrile at room temperature according to the ratio in the above table, sonicate until the two are completely dissolved, and then use a rotary evaporator to evaporate the acetonitrile at 60°C to obtain Transparent gel-like drug-containing lipid film, then quickly add 5-8mL water for injection, vortex, to obtain a transparent polymer micellar solution.

Take 0.5mL of the solution and place it in an ultrafiltration tube ( MWD3000), centrifuged at 10000rpm for 30 minutes, and the obtained ultrafiltrate was diluted with 10 times of acetonitrile, HPLC was injected to analyze the content of docetaxel, and the encapsulation efficiency was calculated.

Then the remaining polymer micelle solution was filtered through a 0.22 μm sterile microporous membrane After filtering, sub-package, freeze in a -70°C refrigerator for 12 hours, and then freeze-dry to prepare polymer freeze-dried micelles encapsulating docetaxel.

Take 1 mg of polymer micelles, dissolve them in 10 ml of acetonitrile, and then dilute them 10 times, analyze the content of docetaxel in HPLC, and calculate the drug loading.

Example 5 Preparation of polymer micelles containing lyoprotectant-loaded docetaxel

Dissolve 10 mg of docetaxel and 50 mg of mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ in 5 mL of acetonitrile at room temperature, sonicate until the two are completely dissolved, and then evaporate the acetonitrile at 60°C using a rotary evaporator to obtain a transparent gel Form a drug film, and then add 5-8mL water for injection at 25°C to obtain a transparent polymer micelle solution. Then add 3 mg PEG ₂₀₀₀ or poloxamer 188 or lactose or glucose or sorbitol or mannitol or mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ or human serum albumin (Human Serum Albumin, HSA) or PVP K30, sterile by 0.22 μm Microporous membrane After filtering, sub-package, freeze in a -70°C refrigerator for 12 hours, and then freeze-dry to prepare docetaxel-loaded polymer lyophilized micelles containing 5% (w/w) lyoprotectant.

Example 6 Preparation of polymer micelles containing lyoprotectant-loaded docetaxel

Dissolve 10mg of docetaxel and 50mg of mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ in 5ml of acetonitrile at room temperature, sonicate until the two are completely dissolved, and then use a rotary evaporator to evaporate the acetonitrile at 60°C to obtain a transparent gel Form a drug film, and then immediately add 5-8ml water for injection to obtain a transparent polymer micelle solution. Then add 6 mg of glucose or mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ or human serum albumin (Human serum albumin, HSA), filter through a 0.22 μm sterile microporous membrane After filtering, sub-package, freeze in a -70°C refrigerator for 12 hours, and then freeze-dry to prepare docetaxel-loaded polymer lyophilized micelles containing 10% (w/w) lyoprotectant.

Experimental Example 1 Test experiment of micelles physical and chemical properties after reconstitution

Freeze-dried micelles of docetaxel of the present invention (note: in experimental examples 1 to 7, unless otherwise specified or otherwise indicated, the block polymers used are all mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ , docetaxel and mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ (mass ratio: 1:5) was stored in a refrigerator at 4°C for half a year, then dissolved in 5% glucose solution, and prepared as a solution with a concentration of 1 mg/ml (calculated as docetaxel).

The particle size of drug-loaded polymer micelles was determined by dynamic light scattering method, and the result was: particle size 18nm, P.I. value 0.129 (Figure 3); meanwhile, transmission electron microscopy was used to further measure the particle size distribution (Figure 4). From the results, the drug-loaded micelles after reconstitution were spherical, with small particle size and uniform distribution.

Experimental Example 2 Stability experiment after reconstitution

The docetaxel lyophilized micelles containing 5% and 10% lyoprotectants prepared according to the present invention were stored in a refrigerator at 4°C for half a year, then dissolved in 5% glucose solution, and prepared at a concentration of 1 mg/ml (in the form of docetaxel) solution.

The particle size of drug-loaded polymer micelles was measured by dynamic light scattering method at 0 hour, 4 hours and 6 hours after reconstitution, and the changing trend is shown in Figure 5.

Considering the appearance of lyophilized powder and the change of particle size after reconstitution, glucose or mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ or HAS are the best choice of lyoprotectant, and the appropriate addition amount is 5%.

Experimental example 3 hemolytic experiment

Take a certain amount of fresh whole blood from 8 male guinea pigs, stir it quickly with a bamboo stick to remove fibrinogen, and make it into defibrinated blood, add 5% glucose injection and centrifuge and wash (1000rpm*15min), repeat several times until the supernatant The obtained erythrocytes were prepared into a 2% (v/v) erythrocyte suspension until the red color did not appear, and the suspension was shaken before use, and freshly prepared during the experiment.

Docetaxel freeze-dried micelles of the present invention (prepared in Example 6) and commercially available docetaxel preparations Use 5% glucose solution to prepare 0.1, 0.25, 0.5 and 0.75 mg/ml solutions respectively, add them to the diluted guinea pig erythrocyte suspension, shake gently and keep them in a water bath at 37°C to keep warm, and take out the test tube at 1 hour , centrifuge (2000rpm*5min), absorb the supernatant and measure the absorbance of each tube solution at 576nm. Calculate the hemolysis value of each test tube sample according to the following formula: Hemolysis %=[(A sample-A negative)/(A positive-A negative)]×100%, the results are as follows:

From the results of in vitro experiments, it can be found that the docetaxel micelles of the present invention have basically no hemolytic effect.

Experimental example 4 hemolytic experiment

The docetaxel freeze-dried micelles of the present invention were stored in a refrigerator at 4°C for half a year, then dissolved in 5% glucose solution, and prepared as a solution with a concentration of 1 mg/ml (calculated as docetaxel). Then the solution was transferred to a dialysis bag (MWD3500), and the dialysis bag was placed in 40ml of phosphate buffered saline (PBS, pH 7.4), and then shaken in an air bath shaker (100rpm, 37°C). Samples were taken at 0.5h, 2h, 4h, 6h, 8h, 12h, 24h, and 36h, and analyzed by HPLC. The release curve shown in Figure 6 was obtained, which confirmed that the micellar preparation of the present invention has a certain sustained release effect.

Experimental example 5MTT experiment

Take 4T1 cells in the logarithmic growth phase, count the diluted cells with a cell counting plate, inoculate 5000 cells/well on a 24-well plate, and grow cells adherently after 6 hours, add samples, and culture for 24 hours, 48 hours and After 72 hours, the cell inhibition rate was detected by MTT assay.

The drug-free culture medium was used as the blank control, and the as a positive control, and a blank micelle without drug loading as a negative control. Prepared with different dosing concentration gradients (0.0001nmol/mL, 0.001nmol/mL, 0.01nmol/mL and 1nmol/mL, calculated as docetaxel), 6 auxiliary wells; the blank micelles were converted to the corresponding excipient concentrations .

As shown in Figure 7, the mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ material is relatively safe, that is, the blank micelles have basically no effect on the inhibitory rate of 4T1 metastatic breast cancer cells, and the inhibitory rates are all less than 3%; docetaxel of the present invention The effect of the micelles on inhibiting tumor cells in vitro was significantly better than that of the commercially available preparations.

Experimental Example 6 Pharmacodynamics experiment for metastatic breast cancer

Tumor cells: mouse metastatic breast cancer cell line 4T1, cultured in RPMI-1640 medium containing 10% fetal bovine serum.

Animals: Balb/c mice, 6 weeks old, female. 5% glucose injection group, 6 mice in each group and docetaxel micelles group.

Tumor cell inoculation: digest 4T1 cells in the logarithmic growth phase under sterile conditions, prepare a cell suspension of 2×105 cells/ml, inoculate 0.1 ml subcutaneously at the fourth mammary pad of each mouse, and follow the steps below after 14 days Table regimen dosing:

Body weight: the mice were weighed before each administration, and the body weight change curve as shown in Figure 8 was drawn.

H&E stained pathological sections: mice were sacrificed on the 26th day after tumor inoculation, lung tissues were taken, fixed in 4% neutral formaldehyde, embedded in paraffin, sectioned, dewaxed, stained, and finally dehydrated and transparently mounted. The H&E staining pictures were obtained as shown in Figure 9 (representative pictures of 6 mice in each group). Compared In the micelles group, no tumor cells were found in the lungs of mice, and the inflammatory response (neutrophil increase) and congestion caused by tumor metastasis were significantly reduced.

It can be seen from Figure 8 and Figure 9 that due to the EPR effect and the small particle size (<25nm) of docetaxel, the docetaxel freeze-dried micelles improved the bioavailability of docetaxel and had a significant therapeutic effect on metastatic breast cancer. Reduced its systemic toxicity.

Experimental Example 7: Pharmacodynamic experiment for pancreatic cancer

Taking the commercially available preparation as a control, the tumor inhibitory effect of the docetaxel micellar preparation of the present invention on orthotopic transplantation of pancreatic cancer was investigated. Female BALB/c Nude15, 5mg/mL pentobarbital sodium solution tail vein injection anesthetized nude mice

(0.3mL/cause), the tumor site was wiped with iodine tincture for disinfection, and the operation was started after deiodination with 75% alcohol later. Use ophthalmic scissors to open the skin and abdominal muscle layer at the left spleen of nude mice, carefully pull out the pancreas from the abdominal cavity, and inoculate 3×10 ⁵ cells/0.02mL of Capan-2 ^luc cell suspension along the direction from the head to the tail of the pancreas , and then the pancreas was backfilled into the abdominal cavity, and the sutures were sterilized. On the 21st day, the nude mice were randomly divided into 6 groups and administered once every 7 days for a total of 3 times: (1) 5% glucose injection group; (2) group, 10 mg/kg was injected into the tail vein; (3) the docetaxel micelles group of the present invention was injected 10 mg/kg into the tail vein. On Day49, the luciferase substrate Luciferin (150 μg/mL) was injected intraperitoneally, nude mice were anesthetized with isoflurane gas for 10-15 minutes, and then the nude mice were fixed in the Xenogen in vivo imaging system to detect the tumor focus in the pancreas of the nude mice bioluminescent condition.

The bioluminescence detection results at day 49 are shown in Figure 10.a. The experimental results show that the tumor transplantation effect of the docetaxel micelle preparation of the present invention is better than that of the commercially available preparation. Figure 10.b is the body weight change curve of nude mice in each group after administration. The body weight of animals in each group did not show a significant downward trend, proving that the 10 mg/kg administration dose was suitable and did not cause large systemic toxicity to animals. The body weight of the animals in the docetaxel micellar preparation group of the present invention was slightly higher than that in the commercially available preparation group.

The present invention is not limited to the above description, and those skilled in the art can make various modifications and variations to the scheme of the present invention without departing from the teaching and gist of the present invention, all of which will be included within the scope of the present invention.

Claims (10)

1. A docetaxel nanomicelle preparation, which comprises antitumor drug docetaxel, amphiphilic block copolymer methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000～1500 , and any Selected pharmaceutically acceptable lyoprotectant. 2. The nanomicelle preparation according to claim 1, wherein the amphiphilic block copolymer methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000～1500 is preferably methyl polyethylene glycol Diol ₂₀₀₀ -b-poly D, L-lactic acid ₁₃₀₀ ; the lyoprotectant is PEG ₂₀₀₀ , poloxamer 188, lactose, glucose, sorbitol, mannitol, mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ , human blood Any one or a mixture of albumin and PVP K30, preferably glucose, mPEG ₂₀₀₀ -b-PDLLA ₁₃₀₀ , human serum albumin. 3. The nanomicelle preparation according to any one of claims 1-2, wherein docetaxel and amphiphilic block copolymer methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000～ The mass ratio of ₁₅₀₀ is 1:3-1:9, preferably 1:5; the added amount of the lyoprotectant is 1-10% based on the mass percentage of the total amount of the lyophilized preparation, preferably 5% by mass. 4. The nanomicelle preparation according to any one of claims 1-3, wherein the micelle preparation is a lyophilized form, and water for injection, 5% glucose injection or 0.9% sodium chloride injection are used as clinical Dispersion medium for medication. 5. The nanomicelle preparation according to claim 1, characterized in that: the drug loading of the nanomicelle preparation is between 10% and 25%, preferably 15% to 18%; the reconstituted nanoparticle The diameter is between 15nm and 25nm, and the dispersion coefficient P.I. of the particles is between 0.09 and 0.15; the encapsulation efficiency of docetaxel in the micelles reaches at least 85%. 6. A method for preparing the docetaxel nano-micelle preparation according to any one of claims 1-5, comprising the following steps: (1) Docetaxel and methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000-1500 are dissolved in an organic solvent; (2) remove the organic solvent to obtain a polymer lipid film containing docetaxel; (3) Add water for injection and hydrate at 25°C to 60°C; (4) Vortex or ultrasonic to obtain a nanomicelle solution; (5) Optionally add a lyoprotectant, filter and sterilize with a 0.22 μm sterile microporous membrane, freeze in a refrigerator at -50°C to -70°C for 12 hours, and then freeze-dry to obtain docetaxel-encapsulated Methyl polyethylene glycol ₂₀₀₀ -b-poly D, L-lactic acid _1000~1500 nanometer micelles preparation. 7. The method according to claim 6, wherein the organic solvent in step (1) is acetonitrile, methanol, ethanol and chloroform, which can be used alone or in combination, preferably acetonitrile. 8. The method according to claim 6 or 7, wherein in step (2) the organic solvent is removed by decompression and/or the organic solvent is removed under vacuum; wherein in step (3) at 25°C to 60°C, preferably Hydrate at 35°C-45°C for 1-2 hours. 9. The method according to any one of claims 6-8, wherein in step (4), vortex shaking or ultrasonication is performed for 1-5 minutes. 10. The application of the nanomicelle formulation according to any one of claims 1-5 in the preparation of a medicament for treating metastatic breast cancer or pancreatic cancer.