CN114533682A - Docetaxel albumin nano composition and preparation method thereof - Google Patents

Abstract

The invention provides a docetaxel albumin nano composition and a preparation method thereof, and particularly provides a docetaxel albumin nano preparation, wherein the preparation comprises the following components in parts by weight: 1-30 parts of docetaxel, 5-50 parts of albumin and 5-85 parts of a stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient.

Description

A kind of docetaxel albumin nanometer composition and preparation method thereof

technical field

The invention belongs to the field of pharmaceutical preparations, relates to a composition or compound used in the field of anti-tumor, and more particularly relates to a preparation method of a nano-composition comprising docetaxel, albumin and stabilizer as a prescription.

Background technique

In recent years, the incidence of tumor has risen sharply, its treatment is difficult and the mortality rate is high, and it has become the second leading cause of human death. According to reports, there were about 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. Among them, there were more than 2.338 million deaths from malignant tumors in China, and the mortality rate exceeded 30%. For every 100 new cancer patients in the world, The Chinese accounted for 21, which has become a serious social problem. At present, the clinical treatment of advanced tumors is mostly combined with radiotherapy and chemotherapy to delay the development of the disease, but all of them are accompanied by severe immune system suppression or toxic side effects, and it is difficult to effectively improve the survival rate and quality of life of patients. Therefore, the search for therapeutic methods or drugs with good curative effect and low toxicity and side effects has always been a research hotspot and an effort direction in the field of tumor therapy.

Docetaxel is a new generation of taxane drugs developed in recent years. Its mechanism of action is similar to that of paclitaxel, but its antitumor activity is 1.3 to 12 times that of paclitaxel. It is more effective than paclitaxel. Although docetaxel has a good anti-tumor effect, the treatment practice shows that it still has the following defects: (1) Docetaxel is almost insoluble in water, and a large amount of surfactant needs to be added to clinical injections, such as commercially available drugs. Docetaxel (Taxotere) contains high concentrations of Tween 80, including the 2015 FDA approval of the non-alcoholic preparation of Eagle Pharmaceuticals. Although it can be stored at room temperature, the stability time after dilution is longer than that of Taxotere, but the prescription contains a large amount of sputum. The serious sensitization side effects caused by Wen-80 in clinical application have not been resolved, such as bone marrow suppression, allergic reactions, neurotoxicity, skin erythema, cardiovascular adverse reactions, etc.; (2) Docetaxel is a cytotoxic drug and does not have Targeted, the drug is rapidly distributed and metabolized in the body after injection, resulting in systemic toxic and side effects. Once overdose occurs in cancer patients after long-term administration, there is no antidote available; (3) Fat emulsion for docetaxel , microemulsion, liposome and other drug delivery systems have not made great progress in reducing adverse reactions and toxic side effects.

To sum up, there is still a lack of a docetaxel pharmaceutical preparation with good curative effect and low toxicity and side effects in the art.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a docetaxel pharmaceutical preparation with good curative effect and low toxic and side effects.

A first aspect of the present invention provides a docetaxel protein nano-formulation, the formulation comprising:

1-30 parts by weight of docetaxel, 5-50 parts by weight of albumin, 5-85 parts by weight of stabilizer; preferably, 1.6-7.2 parts by weight of docetaxel, 10-40 parts by weight of albumin, and stabilizer 50 to 80 parts by weight; and optional pharmaceutical freeze-dried excipients; and the preparation does not include antioxidants;

And in the preparation, docetaxel, albumin and stabilizer together form an albumin nano preparation.

In another preferred embodiment, the average particle size of the nano preparation is 50-150 nm.

In another preferred example, the preparation is freeze-dried powder; preferably, the average particle size of the freeze-dried powder after reconstitution is 50-150 nm.

In another preferred embodiment, the preparation is an injection.

In another preferred embodiment, the injection further includes an optional dispersion medium, and the dispersion medium is selected from the group consisting of sterile water for injection, 5% glucose solution, or physiological saline.

In another preferred example, the docetaxel albumin nanocomposition is 1.6-7.2%, the albumin is 10-40%, the stabilizer is 50-80%, and the sum of the components is 100% , based on the total weight of the three.

In another preferred embodiment, the albumin is selected from the group consisting of recombinant albumin, bovine serum albumin, horse serum albumin, sheep serum albumin, human serum albumin, or a combination thereof; preferably human serum albumin.

In another preferred embodiment, the human serum albumin contains fatty acid.

In another preferred embodiment, the stabilizer is selected from the following group of phospholipids, fatty acids, amino acids, or combinations thereof; preferably phospholipids.

In another preferred example, the phospholipid is selected from the group consisting of egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dilauroyl lecithin, distearoyl lecithin, 1-myristoyl-2-palmitoyl Lecithin, 1-palmitoyl-2-myristoyl lecithin, 1-palmitoyl-2-stearoyl lecithin, 1-stearoyl-2-palmitoyl lecithin, dioleoyl lecithin, dipalmitoyl acylphosphatidylethanolamine, dipalmitoyl glycerol, dipalmitoyl phosphatidic acid, dipalmitoyl sphingomyelin, dipalmitoyl lecithin, dipalmitoyl phosphatidyl disserine, dilauroyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol , dimyristoyl phosphatidic acid, dimyristoyl phosphatidyl ethanolamine, brain phosphatidyl serine, dimyristoyl phosphatidyl lecithin, dimyristoyl phosphatidyl serine, brain sphingomyelin, distearoyl phosphatidyl glycerol, di Stearoyl sphingomyelin or distearoyl phosphatidyl ethanolamine, or a combination thereof.

In another preferred embodiment, the phospholipid is not easy to form liposomes in the preparation.

In another preferred embodiment, the fatty acid is selected from the group consisting of medium chain oil, medium and long chain oil, palm kernel oil, coconut oil, squalene, or a combination thereof.

In another preferred embodiment, the described is at least one selected from the group consisting of arginine, cysteine, lysine, and proline.

In another preferred example, the pharmaceutical freeze-dried auxiliary materials are selected from the following group: sucrose, trehalose, lactose, glucose, mannitol, sorbitol, or a combination thereof; preferably, the pharmaceutical freeze-dried The proportion of excipients is 1-20% (m/m), based on the total mass of the preparation.

In another preferred example, when the pharmaceutical freeze-dried excipients are selected from sucrose, trehalose, lactose, and glucose, the proportion of the pharmaceutical freeze-dried excipients is 5-20% (m/m).

In another preferred example, when the pharmaceutical freeze-dried excipients are selected from mannitol and sorbitol, the proportion of the pharmaceutical freeze-dried excipients is 1-10% (m/m).

In another preferred example, the preparation further includes an organic solvent and/or a dispersion medium; preferably, the organic solvent is selected from the group consisting of dichloromethane, chloroform, acetone, ethyl acetate, ethanol , or a combination thereof; the dispersion medium is selected from the group consisting of water, 5% glucose solution, physiological saline, or a combination thereof.

In another preferred example, the organic solvent is a mixed solvent of ethyl acetate and ethanol, wherein the ratio of ethanol is 10-50% (v/v).

In another preferred embodiment, the preparation includes: 1-3 parts by weight of docetaxel, 15-25 parts by weight of albumin, 70-85 parts by weight of stabilizer; and optional pharmaceutical freeze-dried auxiliary materials; or The preparation comprises: 5-10 parts by weight of docetaxel, 30-40 parts by weight of albumin, 50-70 parts by weight of stabilizer; and optional pharmaceutical freeze-dried auxiliary materials.

In another preferred embodiment, in another preferred embodiment, the preparation comprises: 1.5-2 parts by weight of docetaxel, 18-22 parts by weight of albumin, 75-80 parts by weight of stabilizer; and optional Pharmaceutical freeze-dried auxiliary materials; or the preparation comprises: 6-8 weight parts of docetaxel, 30-35 weight parts of albumin, 55-65 weight parts of stabilizer; and optional pharmaceutical freeze-dried auxiliary materials.

The second aspect of the present invention provides a preparation method of the nano-formulation as described in the first aspect of the present invention, the method comprising the steps of:

(1) according to the formula, provide docetaxel albumin nano-composition, albumin and stabilizer, and optional pharmaceutical freeze-dried adjuvant, organic solvent and dispersion medium;

(2) the docetaxel albumin nano-composition and the stabilizer are dispersed in an organic solvent to form an oil phase, and the albumin is dissolved in an appropriate amount of deionized water to form an aqueous phase;

(3) the water phase is placed in a high-pressure homogenizer or a milk homogenizer for high shear stirring, and at -10 to 40 ° C, the oil phase is injected into the water phase for emulsification while high shear stirring, to form an emulsion;

(4) carrying out high pressure homogenization treatment to the emulsion to obtain a nanoparticle emulsion;

(5) performing decompression rotation or thin film evaporation on the emulsion to remove the organic solvent to obtain a docetaxel-albumin nanocomposite.

In another preferred embodiment, the method is an emulsification method.

In another preferred example, the high shear stirring speed in the step (3) is 500-10000 rpm; and/or

The emulsification time in the step (3) is 2～30min; and/or

In another preferred embodiment, the step (2) is completed at a temperature of 0°C to 25°C.

In another preferred embodiment, the step (3) is completed at a temperature of 0°C to 25°C.

In another preferred embodiment, the high pressure homogenization is completed at a temperature of 0°C to 15°C.

In another preferred embodiment, in the step (5), the decompression rotation or thin film evaporation is completed at a temperature of 25°C to 60°C.

In another preferred embodiment, in the step (5), the decompression rotation or the thin film evaporation rotation speed is completed in the range of 20 rpm to about 400 rpm.

In another preferred embodiment, in the step (5), the decompression rotation is completed under a vacuum degree of 0.1 MPa to about 0.06 MPa.

In another preferred example, the method further comprises: performing ultrasonic or high pressure homogenization process on the docetaxel-albumin nanocomposite, thereby obtaining a nanocomposite with uniform particle size.

In the high pressure homogenization process, the pressure is 5000-30000 psi.

In another preferred example, in the high-pressure homogenization process, the homogenization time is 5-60 min.

In another preferred example, the method further comprises: freeze-drying the obtained docetaxel-albumin nanocomposite; preferably, the freeze-drying comprises the steps:

mixing the docetaxel-albumin nanocomposite with pharmaceutical freeze-dried excipients, and then pre-freezing at -50 to -30° C. for 4 hours;

Perform one sublimation at 0.01-0.03mbar (preferably, the sublimation includes: -35～-25°C for 48h, and then -10～0°C for 12h);

Perform secondary sublimation (preferably for 6 h) at 20-30° C. to obtain a freeze-dried product.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the transmission electron microscope image of the prepared docetaxel albumin nanocomposite composition and the particle size distribution image placed at room temperature for 24h;

Figure 2. Stability of docetaxel albumin nanoparticles in 5% HSA simulated plasma. A. 1 μg/ml; B. 10 μg/ml; C. 100 μg/ml;

Figure 3 Drug release of commercially available docetaxel injection and docetaxel albumin nanoparticles. INJ.: Commercially available injection; HSA NP: Docetaxel albumin nanoparticles;

Figure 4 shows the tumor volume and time of human non-small cell lung cancer A549 after administration of docetaxel albumin nanocomposite low-dose (DNPL), high-dose (DNPH) formulation, DTX injection and blank group of the present invention relation;

Figure 5 shows the change of body weight with time after administration of docetaxel albumin nanocomposite or docetaxel injection to normal mice of the present invention;

Figure 6 shows the relationship between the cytotoxicity of the DTX nanocomposite group and the solution in vitro human non-small cell lung cancer A549 cytotoxicity and time in the present invention;

Figure 7 shows the appearance of the lyophilized cake (left) and the liquid (right) after reconstitution of the docetaxel albumin nanocomposite lyophilized preparation of the present invention before reconstitution.

Detailed ways

The purpose of the present invention is to overcome the defects of the existing preparations and the redundancy of the prescription, and at the same time overcome the poor stability of the current docetaxel albumin nanoparticles, and fill in the blank of the unmonitored docetaxel related substances, protein dimers and lysophospholipids , to provide a stable docetaxel albumin nanometer composition, and the prescription and preparation process can be industrialized and enlarged. In the present invention, the poorly soluble docetaxel drug is prepared as a tumor-targeted albumin nano-composition for injection, which has higher drug loading, good tumor-targeting effect and sustained-release effect compared with the existing preparation, and The preparation process is simple and convenient, and is suitable for industrialized large-scale production.

Docetaxel-albumin nanocomposite

In the soluble albumin nano-composition for injection of the present invention, the docetaxel drug and the stabilizer are combined with albumin and dispersed and stabilized. The present invention utilizes the characteristics of high binding rate between docetaxel and albumin, so that the two are naturally formed and made into albumin nanoparticles, the stabilizer can solubilize and disperse the insoluble drugs, and finally soluble albumin for injection is prepared Nanocomposition.

The essential difference between the formulation of the present invention and the prior art is that the stabilizer is used as a medium for dispersing and stabilizing the complex of the drug and albumin, and together with the drug and albumin, a stable nano-formulation is formed, wherein the phospholipid does not form lipid alone. plastid.

Specifically, the docetaxel drug albumin nano-composition for injection of the present invention is characterized in that, it is made of docetaxel drug, albumin, stabilizer and pharmacy necessary auxiliary materials, wherein, docetaxel drug The mass ratios of the drug, albumin and stabilizer are respectively 1-30%, 5-50% and 5-85%, and the sum of the components is 100%. Preferably, the mass ratios of the docetaxel drug, albumin and phospholipid are 1.6-7.2%, 10-40%, and 50-80%, respectively.

In the present invention, the preferred nano-albumin composition comprises: 1-3 wt % of docetaxel, 15-25 wt % of albumin, 70-85 wt % of stabilizer; and optional pharmaceutical freeze-dried adjuvants; or the described The preparation comprises: docetaxel 5-10wt%, albumin 30-40wt%, stabilizer 50-70wt%; and optional pharmaceutical freeze-dried auxiliary materials. In a more preferred embodiment, the albumin nanocomposition includes: 1.5-2wt% docetaxel, 18-22wt% albumin, 75-80wt% stabilizer; and optional pharmaceutical freeze-dried excipients or the preparation comprises: docetaxel 6-8wt%, albumin 30-35wt%, stabilizer 55-65wt%; and optional pharmaceutical freeze-dried auxiliary materials.

In the present invention, the albumin is human serum albumin or bovine serum albumin, but preferably human serum albumin.

In the present invention, the phospholipid is not particularly limited, and can be selected from (but not limited to): egg yolk lecithin (EPC), soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), dilauroyl lecithin (DLPC) ), distearoyl lecithin (DSPC), 1-myristoyl-2-palmitoyl lecithin (MPPC), 1-palmitoyl-2-myristoyl lecithin (PMPC), 1-palmitoyl-2 - Stearoyl lecithin (PSPC), 1-stearoyl-2-palmitoyl lecithin (SPPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dipalmitoyl glycerol ( DPPG), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl sphingomyelin (DPSP), dipalmitoyl lecithin (DPPC), dipalmitoyl phosphatidyl disserine (DPPS), dilauroyl phosphatidyl glycerol (DLPG) ), dioleoyl phosphatidyl glycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dimyristoyl phosphatidyl ethanolamine (DMPE), brain phosphatidyl serine (PS), dimyristoyl lecithin (DMPC) , dimyristoyl phosphatidyl serine (DMPS), brain sphingomyelin (BSP), distearoyl phosphatidyl glycerol (DSPG), distearoyl sphingomyelin (DSSP) or distearoyl phosphatidyl ethanolamine ( One or more of DSPE); preferably EPC, SPC, DSPE or HSPC.

The docetaxel albumin nanometer composition prepared by the invention can be stable for more than 24 hours at room temperature, and can be stable for more than 72 hours at 4°C, and does not cause docetaxel related substances, stabilizer related substances and protein dimerization Excessive body increase, among which, the related substances of docetaxel only increased by 0.04%, and the protein dimer only increased by 1.45%. The peroxide value of stabilizers such as phospholipids, free fatty acids, LPE and LPC are all within the requirements of the Pharmacopoeia, which is beneficial to Industrialization scales up production. The prepared lyophilized powder injection has good appearance, fast reconstitution speed, little difference between the properties after reconstitution and the liquid before lyophilization, and can still be stable for 24 hours at room temperature and 4°C, which greatly facilitates clinical application.

Preparation of docetaxel-albumin nanocomposite

The present invention adopts the emulsification method to prepare the albumin nanometer composition for injection, and can selectively adopt ultrasonic or high pressure homogenization process to further control the particle size. In one embodiment of the present invention, docetaxel in a given prescription is dissolved in an appropriate amount of ethanol or other suitable solvent, and stabilizers such as phospholipids and fatty acids are dissolved in an appropriate amount of dichloromethane, and the two are mixed as an oil phase ; Dissolve albumin in an appropriate amount of deionized water (water phase), drop the oil phase into the magnetically stirred water phase at room temperature or in an ice-water bath, and continue to stir for a certain period of time, and then remove ethanol and Dichloromethane to prepare docetaxel-loaded albumin nanocomposites.

In the present invention, after the docetaxel drug is prepared into albumin nanoparticles, its dispersion medium can be water, 5% glucose solution or physiological saline.

Docetaxel-albumin nanocomposite preparation

Due to its good pharmacokinetics, the soluble albumin nano-composition for injection of the present invention can be used to prepare a drug for treating cancer, or be used as a drug for cancer treatment; wherein, the cancer includes lung cancer, breast cancer, and ovarian cancer , brain glioma, liver cancer, pancreatic ductal cancer, esophageal cancer, gastric cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, endometrial cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer , testicular cancer, skin cancer, lymphoma, head and neck tumors and primary or secondary carcinomas, sarcomas or carcinomas originating in the gallbladder, oral cavity, peripheral nervous system, mucous membranes, glands, blood vessels, bone tissue, lymph nodes, eyes Sarcoma, etc.

In the present invention, the soluble albumin nano-composition for injection can be administered by intravenous, subcutaneous, intramuscular, intramembranous, intraperitoneal or other routes.

In the present invention, after the docetaxel drug is encapsulated, the dose of docetaxel drug is 0.01-20 mg/kg each time, preferably 1-10 mg/kg per administration; The scheme is daily administration or interval administration, and the dosage for each course of treatment is 0.3-600 mg/kg, and the preferred dosage for each course of treatment is 4-40 mg/kg.

The pharmacodynamic test of the soluble albumin nano-composition for injection of the present invention shows that its therapeutic effect is better than that of the commercially available injection.

The present invention utilizes the enhanced penetration and retention effect (EPR effect) of nanoparticles on tumors, so that more drugs are passively targeted and concentrated in tumor tissue, thereby improving the anti-tumor effect; at the same time, due to the high bioavailability of the injection method and the The soluble albumin nano-composition for injection has a passive targeting effect, which can greatly reduce the dosage, thereby effectively reducing the drug concentration in non-target sites, and helping to reduce the toxic and side effects of the drug; The protein nano-composition avoids the shortcomings of low bioavailability and poor patient compliance of marketed oral preparations, and combines the water-solubility-improving effect and passive targeting effect of albumin nanoparticles, which has a good clinical application prospect. On the other hand, the preparation method of the present invention is simple, the drug recovery rate is high, and is suitable for large-scale industrial production.

Compared with the prior art, the soluble albumin nano-composition for injection of the present invention has the following remarkable advantages:

①It has a significant solubilizing effect on docetaxel, and its solubility is sufficient for clinical injection;

② The encapsulation rate is almost 100%, there is no loss in the preparation process, the recovery rate is higher than 85%, and the drug loading is also high;

③The soluble albumin nanocomposition for injection can utilize the EPR effect of the tumor site and the active targeting effect of SPARC-gp60 to increase the accumulation of the drug in the tumor tissue, which is beneficial to exert the anti-tumor effect of the drug and reduce the toxic and side effects on other tissues.

④It has a slow-release effect, which can continuously kill tumor cells and reduce the number of administrations.

⑤ The preparation is stable, stable for more than 24 hours at room temperature, and stable for more than 72 hours at 4 °C, and will not cause excessive increase in docetaxel related substances, lysophospholipids and protein dimers, which is conducive to mass production;

⑥ The prepared lyophilized powder for injection has good appearance and fast reconstitution speed. The properties after reconstitution are very small from those before lyophilization, and can still be stable for 24 hours at room temperature and 4°C, which greatly facilitates clinical application.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1: Prescription and preparation process of docetaxel albumin nanocomposite

Table 1 Proportion (mg) of each component in the formulation of docetaxel-encapsulated albumin nanocomposites

Dissolve docetaxel in prescription F01-20 in Table 1 in an appropriate amount of chloroform or other suitable solvent, dissolve the stabilizer in an appropriate amount of ethanol, and mix the two as an oil phase; dissolve albumin in an appropriate amount of deionized water. In water (aqueous phase), drop the oil phase into the water phase at room temperature or in an ice-water bath, stir and disperse the initial emulsification for a certain period of time, and then carry out nano-dispersion in a high-pressure homogenizer or an emulsion homogenizer, and then perform rotary evaporation or thin film in a water bath at 50°C. The organic solvent is removed by evaporation to obtain an albumin nanocomposition comprising docetaxel.

The results obtained are shown in Table 2. The average particle size of the obtained preparation varies with different components, and at 50-150 nm, the recovery rate is all above 85%, and the encapsulation rate is all above 98%. Since the stability of the preparation prepared by the present invention cannot be reflected by the particle size, in the previous formulation screening process, the preparation had precipitation visible to the naked eye, but the particle size did not change significantly. Therefore, we use the turbidity change rate to measure the stability of the preparation (See Note in Table 2 for the specific calculation method). When there is drug precipitation, the turbidity change rate is very large, and when there is no drug precipitation, the turbidity change rate fluctuates within a limited range, basically within 10%.

It can be seen from Table 2 that the preparations prepared by the prescriptions in the table can be stable for more than 24h at room temperature, some prescriptions can even be stable for 48h, and at 4°C, they can be stable for more than 72h.

Table 2 Results of different proportions of component preparations

Note: Turbidity change rate=(A _T -A ₀ )/A ₀ *100%, A ₀ represents the absorbance of the nanosuspension at 350 nm at T0, and A _T represents the absorbance at T hours.

Example 2: Characterization of Docetaxel Albumin Nanocomposition - Morphology and Particle Size

A small amount of albumin nanocomposites were taken on a copper mesh, negatively stained with phosphotungstic acid, and the morphology of the nanocomposites was observed under a transmission electron microscope. The particle size of the prepared docetaxel albumin nanocomposites was measured using a dynamic light scattering particle size analyzer, and the average particle size was measured to be 132.4±0.59 nm. The results of electron microscopy and particle size distribution are shown in Figure 1, respectively. The results show that the nano-composition of the present invention is spherical, and the appearance is round and relatively uniform.

Example 3: In vitro stability assay

(1) Experimental method

Dilute HSA (20%) to 5% with PBS, adjust the temperature of the particle sizer to 37°C, and dilute the drug with a solution of 5% HSA to the drug concentration of 1, 10, and 100 μg/ml. The 0, 10, 20, 30, 60min detection particle size.

(2) Experimental results

The experimental results are shown in Figure 2. In the case of low concentration, the nanoparticles dissociated quickly to form a drug-albumin complex, which is more conducive to the penetration of the drug in the tumor site.

Example 4 Drug release experiment

(1) Experimental method

Dilute the docetaxel injection and docetaxel albumin nanocomposite to 1mg/ml, respectively take 1ml and add it to a dialysis bag with a molecular weight of 10000kDa, and place the dialysis bag in PBS with pH=5.0 and pH=7.4, respectively. Each group of 3 parts was placed on a shaker (37°C, 100rpm) and 1ml was taken at 0.1, 0.25, 0.5, 1, 2, 4, 8, 16, 2, 48, and 72 hours for testing. After each sampling, 1ml was added. Fresh release medium.

(2) Experimental results

The experimental results are shown in Fig. 3. The injection has drug precipitation after 2 hours, so the drug has no sudden release effect. There was little difference in the release of the injections at the two pHs. Albumin nanoparticles were released relatively completely under acidic conditions, up to 91%.

Example 5: Pharmacodynamic test of docetaxel-encapsulated albumin nanocomposites on subcutaneous non-small cell lung cancer

Tumor cells: human non-small cell lung cancer A549 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences), cultured in DMEM medium containing 10% fetal bovine serum.

Animals: Balb/c nude mice, 4 weeks old, male. The experimental group and the negative control group consisted of 6 nude mice each. Inoculation of tumor cells: A subcutaneous inoculation model was used in the right axilla. The vigorously growing A549 cells were taken under sterile conditions, the cells were digested and centrifuged and resuspended into a cell suspension of about 2.0E7/ml, and 0.1 0.1 was subcutaneously inoculated into the right armpit of each mouse. ml cancer cell suspension.

The calculation methods of tumor volume and tumor inhibition rate were the same as before. Each group was administered according to the dosing schedule in Table 3 for two weeks, and the tumor inhibition effects were shown in Figures 4-6. The results show,

Table 3 Docetaxel albumin nanocomposites on subcutaneous non-small cell lung cancer model mice pharmacodynamics tumor inhibitory experiment dosing schedule

DTX injection i.v., 10mg/kg, once every three days DNP low dose i.v., 5mg/kg, once every three days DNP high dose i.v., 10mg/kg, once every three days control group i.v., 0.9%NaCl

Table 4 shows the pharmacokinetic parameters of the docetaxel albumin nanocomposition and the marketed solution in the present invention after administration to rats.

Table 4 Pharmacokinetic parameters of docetaxel injection (commercially available) and docetaxel albumin nanocomposites

The results show that the docetaxel nano preparation of the present invention can effectively improve the pharmacokinetics after injection and increase the utilization rate of the drug in vivo.

Example 6: Encapsulation of docetaxel albumin nanocomposite plus lyophilized support agent

The docetaxel-loaded albumin nanocomposite solution is added with a freeze-drying protective agent (trehalose, sucrose or mannitol) for freeze-drying, and the photo of its properties is shown in Figure 7. The surface of the freeze-dried powder is smooth and fine, and the uniform white cake , plump, no obvious collapse and cracks; reconstituted rapidly, the reconstituted solution is translucent, the particle size is less than 150nm and the distribution is uniform.

Example 7: Solvent Residual

1. Experimental method

(1) Weigh the lyophilized powder, then reconstitute the lyophilized powder to 10ml.

(2) Take a 2ml to 20ml headspace bottle, and then detect the content of ethanol and dichloromethane on a gas chromatograph, and calculate the residual solvent ratio.

2. Experimental results

The results of solvent residues in freeze-dried products are shown in Table 7. It can be seen from the table that both ethanol and dichloromethane of the final products meet the requirements of the pharmacopoeia (ethanol is not higher than 0.5%; dichloromethane is not higher than 0.06%).

Table 5 Solvent residues in freeze-dried products

Example 8: Moisture determination

1. Experimental method

Weigh the freeze-dried powder, then put the freeze-dried powder in the titration bottle of the moisture analyzer, input the mass of the freeze-dried powder, and the moisture analyzer will automatically calculate the moisture content of the freeze-dried powder.

2. Experimental results

The moisture content of freeze-dried products is shown in Table 6, and the moisture content of freeze-dried products meets the quality requirements.

Table 6 Freeze-dried product moisture

Example 9: Formulation Impurity Determination

1. Experimental method

(1) Reconstitute the lyophilized powder to 10ml.

(2) Take 1ml of the reconstituted preparation into a 10ml volumetric flask, add 2ml of ethanol and 2ml of acetonitrile, sonicate for 5min, and then dilute to 10ml with a diluent (water:acetonitrile:acetic acid=50:50:0.5).

(3) Centrifuge at 12,000 rpm for 20 min, and take the supernatant into a liquid chromatograph for detection.

2. Experimental results

The results of related substances of docetaxel are shown in Table 7, and the related substances of the prepared preparation only increased by 0.04%.

Table 7 Docetaxel related substance ratio

Example 10: Protein Dimers

1. Experimental method

(1) Reconstitute the lyophilized powder to 10ml.

(2) Take 1ml of the reconstituted preparation into a 10ml volumetric flask, add ultrapure water to make the volume to 10ml, mix well and then enter the liquid chromatograph for detection.

2. Experimental results

The results of protein dimerization are shown in Table 8, and the related substances of the prepared preparation only increased by 1.45%.

Table 8 Determination results of protein dimer content of docetaxel albumin nanocomposites

Example 11: Phospholipid related detection

1. Experimental method

According to the Chinese Pharmacopoeia 2015 edition, the fourth part of egg lecithin (for injection) method detection.

2. Experimental results

The phospholipid-related detection results in the docetaxel albumin nanocomposites are shown in Table 9. It can be seen from the table that the contents of lipid peroxides, free fatty acids, LPC and LPE all meet the standards of the National Pharmacopoeia.

Table 9 Detection of phospholipids in docetaxel albumin nanocomposites

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A docetaxel protein nano-formulation, comprising:

1-30 parts of docetaxel, 5-50 parts of albumin and 5-85 parts of a stabilizer; preferably, 1.6 to 7.2 parts by weight of docetaxel, 10 to 40 parts by weight of albumin, and 50 to 80 parts by weight of stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient; and said formulation does not include an antioxidant;

in the preparation, docetaxel, albumin and a stabilizer form an albumin nano preparation together.

2. The NanoPreformulation according to claim 1, wherein said albumin is selected from the group consisting of: recombinant albumin, bovine serum albumin, horse serum protein, sheep serum protein, human serum albumin, or a combination thereof; human serum albumin is preferred.

3. The nanoformulation according to claim 1, wherein the stabilizer is selected from the group consisting of phospholipids, fatty acids, amino acids, or combinations thereof; phospholipids are preferred.

4. The nano-formulation of claim 1, wherein the lyophilized pharmaceutical excipients are selected from the group consisting of: sucrose, trehalose, lactose, glucose, mannitol, sorbitol, or a combination thereof; preferably, the proportion of the medicinal freeze-drying auxiliary materials is 1-20% (m/m) based on the total mass of the preparation.

5. The nano-formulation according to claim 1, wherein the formulation further comprises an organic vehicle and/or a dispersion medium; preferably, the organic solvent is selected from the group consisting of: dichloromethane, chloroform, acetone, ethyl acetate, ethanol, or combinations thereof; the dispersion medium is selected from the group consisting of: water, a 5% glucose solution, physiological saline, or a combination thereof.

6. The nano-formulation of claim 1, wherein the formulation comprises: 1-3 parts of docetaxel, 15-25 parts of albumin and 70-85 parts of a stabilizer; and optionally a pharmaceutical lyophilization excipient; or the formulation comprises: 5-10 parts of docetaxel, 30-40 parts of albumin and 50-70 parts of a stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient.

7. The nano-formulation according to claim 1, wherein in another preferred embodiment, the formulation comprises: 1.5-2 parts of docetaxel, 18-22 parts of albumin and 75-80 parts of stabilizer; and optionally a pharmaceutical lyophilization excipient; or the formulation comprises: 6-8 parts of docetaxel, 30-35 parts of albumin and 55-65 parts of a stabilizer; and optionally a pharmaceutical lyophilization excipient.

8. The method for preparing a nano formulation according to claim 1, comprising the steps of:

(1) according to the formula, a docetaxel albumin nano composition, albumin, a stabilizer, optional medicinal freeze-drying auxiliary materials, an organic solvent and a dispersion medium are provided;

(2) dispersing the docetaxel albumin nano composition and a stabilizer in an organic solvent to form an oil phase, and dissolving albumin in a proper amount of deionized water to form a water phase;

(3) placing the water phase in a high-pressure homogenizer or an emulsion homogenizer for high-shear stirring, and injecting the oil phase into the water phase for emulsification at the temperature of minus 10-40 ℃ while carrying out high-shear stirring, thereby forming an emulsion;

(4) carrying out high-pressure homogenization treatment on the emulsion to obtain nano-particle emulsion;

(5) and carrying out reduced pressure rotation or film evaporation on the emulsion to remove the organic solvent, thereby obtaining the docetaxel-albumin nano composition.

9. The method according to claim 8, wherein the high shear stirring speed in the step (3) is 500-10000 rpm; and/or

The emulsifying time in the step (3) is 2-30 min; and/or

In the high-pressure homogenizing process, the pressure is 5000-30000 psi.

10. The method of claim 8, wherein the method further comprises: carrying out freeze-drying treatment on the obtained docetaxel-albumin nano composition; preferably, the lyophilization process comprises the steps of:

mixing the docetaxel-albumin nano composition with a medicinal freeze-drying auxiliary material, and then pre-freezing for 4 hours at-50 to-30 ℃;

performing sublimation once at 0.01-0.03mbar (preferably, the sublimation comprises maintaining at-35 deg.C to-25 deg.C for 48h, and then maintaining at-10 deg.C to 0 deg.C for 12 h);

carrying out secondary sublimation (preferably 6 hours) at 20-30 ℃ to obtain a freeze-dried product.

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