A docetaxel albumin nano-composition and a method of preparing the same

Abstract

The present application provides a docetaxel albumin nano-composition and a preparation method thereof. Specifically, the present application provides a docetaxel albumin nano-preparation, which comprises: 1-30 parts by weight of docetaxel, 5-50 parts by weight of albumin, 5-85 parts by weight of a stabilizer; and optionally a pharmaceutical lyophilized excipient.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations and relates to compositions or complexes for use in the field of antitumor therapy, and more specifically to a method for preparing nanocompositions containing docetaxel, albumin and stabilizers as formulations. Background Technology

[0002] In recent years, the incidence of cancer has risen sharply. Its treatment is challenging and its mortality rate is high, making it the second leading cause of death worldwide. Reports indicate that in 2018, there were approximately 18.1 million new cancer cases and 9.6 million cancer deaths globally. Of these, over 2.338 million deaths were from malignant tumors in China, a mortality rate exceeding 30%. Globally, 21 out of every 100 new cancer patients are Chinese, making it a serious social problem. Currently, clinical treatment for advanced cancer often involves a combination of radiotherapy and chemotherapy to slow disease progression. However, both methods are accompanied by severe immune system suppression or toxic side effects, making it difficult to effectively improve patients' survival rates and quality of life. Therefore, exploring treatment methods or drugs with good efficacy and lower toxicity has always been a research hotspot and a key focus in the field of cancer treatment.

[0003] 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 has a higher efficacy rate than paclitaxel in the treatment of metastatic breast cancer and non-small cell lung cancer. Although docetaxel has good anti-tumor effects, treatment practice shows that it still has the following defects: (1) Docetaxel is almost insoluble in water, and the injectable formulations used in clinical practice all require the addition of a large amount of surfactant. For example, commercially available docetaxel (Taxotere) contains a high concentration of Tween 80. Including the alcohol-free formulation of Eagle Pharmaceuticals approved by the FDA in 2015, although it can be stored at room temperature and has a longer stability time after dilution than Taxotere, the large amount of Tween-80 in the prescription has led to serious sensitization side effects in clinical applications that have not yet been resolved, such as bone marrow suppression, allergic reactions, neurotoxicity, skin erythema, and cardiovascular adverse reactions; (2) Docetaxel is a cytotoxic drug and does not have a targeting function. After the injection is administered, the drug is rapidly distributed and metabolized throughout the body, producing systemic toxic side effects. If an overdose occurs in a cancer patient after long-term administration, there is no antidote available; (3) There has been no significant progress in reducing adverse reactions and toxic side effects of docetaxel through fat emulsions, microemulsions, liposomes and other drug delivery systems.

[0004] In summary, there is currently a lack of a docetaxel formulation with good efficacy and low toxicity in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a docetaxel drug formulation with good efficacy and low toxicity.

[0006] In a first aspect, the present invention provides a docetaxel protein nanoformulation, the formulation comprising:

[0007] The formulation comprises 1-30 parts by weight of docetaxel, 5-50 parts by weight of albumin, and 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 50-80 parts by weight of stabilizer; and optionally, pharmaceutical lyophilized excipients; and the formulation does not include antioxidants.

[0008] Furthermore, in the aforementioned formulation, docetaxel, albumin, and stabilizer together form an albumin nano-formulation.

[0009] In another preferred embodiment, the average particle size of the nanoformulation is 50-150 nm.

[0010] In another preferred embodiment, the formulation is a lyophilized powder; preferably, the average particle size of the lyophilized powder after reconstitution is 50-150 nm.

[0011] In another preferred embodiment, the preparation is an injection.

[0012] In another preferred embodiment, the injection further includes an optional dispersion medium selected from the group consisting of sterile water for injection, 5% glucose solution, or physiological saline.

[0013] In another preferred embodiment, the docetaxel albumin nanocomposite comprises 1.6-7.2%, albumin comprises 10-40%, and stabilizer comprises 50-80%, and the sum of all components is 100% by total weight of the three.

[0014] 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 combinations thereof; preferably human serum albumin.

[0015] In another preferred embodiment, the human serum albumin contains fatty acids.

[0016] In another preferred embodiment, the stabilizer is selected from the group consisting of phospholipids, fatty acids, amino acids, or combinations thereof; phospholipids are preferred.

[0017] In another preferred embodiment, the phospholipid is selected from the group consisting of: egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dilauroyl lecithin, distearyl lecithin, 1-myristoyl-2-palmitoyl lecithin, 1-palmitoyl-2-myristoyl lecithin, 1-palmitoyl-2-stearoyl lecithin, 1-stearoyl-2-palmitoyl lecithin, dioleoyl lecithin, dipalmitoylphosphatidylethanolamine, dipalmitoylglycerol, and dipalmitoylphosphatidylphosphatidylethanolamine. Fatty acid, dipalmitoyl sphingomyelin, dipalmitoyl lecithin, dipalmitoyl phosphatidyl diserine, dilauroyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dimyristoyl phosphatidyl ethanolamine, cerebrophosphatidyl serine, dimyristoyl lecithin, dimyristoyl phosphatidyl serine, cerebrosphingomyelin, distearyl phosphatidyl glycerol, distearyl sphingomyelin or distearyl phosphatidyl ethanolamine, or combinations thereof.

[0018] In another preferred embodiment, the phospholipids do not readily form liposomes in the formulation.

[0019] In another preferred embodiment, the fatty acid is selected from the group consisting of medium-chain oils, medium- and long-chain oils, palm kernel oil, coconut oil, squalene, or combinations thereof.

[0020] In another preferred embodiment, the ingredient is selected from at least one of the following: arginine, cysteine, lysine, and proline.

[0021] In another preferred embodiment, the pharmaceutical freeze-dried excipient is selected from the group consisting of sucrose, trehalose, lactose, glucose, mannitol, sorbitol, or combinations thereof; preferably, the proportion of the pharmaceutical freeze-dried excipient is 1-20% (m / m) based on the total mass of the preparation.

[0022] In another preferred embodiment, when the pharmaceutical freeze-dried excipient is selected from sucrose, trehalose, lactose, or glucose, the proportion of the pharmaceutical freeze-dried excipient is 5-20% (m / m).

[0023] In another preferred embodiment, when the pharmaceutical lyophilized excipient is selected from mannitol or sorbitol, the proportion of the pharmaceutical lyophilized excipient is 1-10% (m / m).

[0024] In another preferred embodiment, the formulation 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 combinations thereof; and the dispersion medium is selected from the group consisting of water, 5% glucose solution, physiological saline, or combinations thereof.

[0025] In another preferred embodiment, the organic solvent is a mixture of ethyl acetate and ethanol, wherein the proportion of ethanol is 10-50% (v / v).

[0026] In another preferred embodiment, the formulation comprises: 1-3 parts by weight of docetaxel, 15-25 parts by weight of albumin, 70-85 parts by weight of stabilizer; and optional lyophilized pharmaceutical excipients; or the formulation comprises: 5-10 parts by weight of docetaxel, 30-40 parts by weight of albumin, 50-70 parts by weight of stabilizer; and optional lyophilized pharmaceutical excipients.

[0027] In another preferred embodiment, the formulation comprises: 1.5 to 2 parts by weight of docetaxel, 18 to 22 parts by weight of albumin, 75 to 80 parts by weight of stabilizer; and optional lyophilized pharmaceutical excipients; or the formulation comprises: 6 to 8 parts by weight of docetaxel, 30 to 35 parts by weight of albumin, 55 to 65 parts by weight of stabilizer; and optional lyophilized pharmaceutical excipients.

[0028] A second aspect of the present invention provides a method for preparing nano-formulations as described in the first aspect of the present invention, the method comprising the steps of:

[0029] (1) Provided, according to the formulation, a docetaxel albumin nanocomposition, albumin and stabilizer, and optional pharmaceutical lyophilized excipients, organic solvents and dispersion media;

[0030] (2) The docetaxel albumin nanocomposition and stabilizer are dispersed in an organic solvent to form an oil phase, and albumin is dissolved in an appropriate amount of deionized water to form an aqueous phase;

[0031] (3) The aqueous phase is placed in a high-pressure homogenizer or emulsifier for high-shear stirring. At -10 to 40°C, the oil phase is injected into the aqueous phase for emulsification while being stirred under high shear, thereby forming an emulsion.

[0032] (4) The emulsion is subjected to high-pressure homogenization to obtain a nanoparticle emulsion;

[0033] (5) The organic solvent is removed by depressurization rotation or thin film evaporation of the emulsion to obtain docetaxel-albumin nanocomposition.

[0034] In another preferred embodiment, the method is an emulsification method.

[0035] In another preferred embodiment, the high-shear stirring speed in step (3) is 500–10000 rpm; and / or

[0036] The emulsification time in step (3) is 2–30 min; and / or

[0037] In another preferred embodiment, step (2) is performed at a temperature of 0°C to 25°C.

[0038] In another preferred embodiment, step (3) is performed at a temperature of 0°C to 25°C.

[0039] In another preferred embodiment, the high-pressure homogenization is performed at a temperature of 0°C to 15°C.

[0040] In another preferred embodiment, in step (5), the reduced pressure rotation or thin film evaporation is performed at a temperature of 25°C to 60°C.

[0041] In another preferred embodiment, in step (5), the reduced pressure rotation or thin film evaporation speed is in the range of 20 rpm to about 400 rpm.

[0042] In another preferred embodiment, in step (5), the decompression rotation is performed at a vacuum of 0.1 MPa to about 0.06 MPa.

[0043] In another preferred embodiment, the method further includes subjecting the docetaxel-albumin nanocomposition to an ultrasonic or high-pressure homogenization process to obtain a nanocomposition with uniform particle size.

[0044] In the high-pressure homogenization process, the pressure is 5000-30000 psi.

[0045] In another preferred embodiment, the homogenization time in the high-pressure homogenization process is 5 to 60 minutes.

[0046] In another preferred embodiment, the method further includes: lyophilizing the obtained docetaxel-albumin nanocomposition; preferably, the lyophilization process includes the step of:

[0047] The docetaxel-albumin nanocomposite was mixed with pharmaceutical lyophilized excipients and then pre-frozen at -50 to -30°C for 4 hours.

[0048] A sublimation is performed at 0.01-0.03 mbar (preferably, the sublimation includes: maintaining at -35 to -25°C for 48 hours, and then maintaining at -10 to 0°C for 12 hours);

[0049] The product is subjected to a second sublimation at 20–30°C (preferably for 6 hours) to obtain the freeze-dried product.

[0050] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0051] Figure 1 The transmission electron microscope image of the prepared docetaxel albumin nanocomposite and the particle size distribution after 24 hours of storage at room temperature are shown.

[0052] Figure 2 Stability of docetaxel albumin nanoparticles in 5% HSA-mimicking plasma. A. 1 μg / ml; B. 10 μg / ml; C. 100 μg / ml;

[0053] Figure 3 Drug release profiles of commercially available docetaxel injection and docetaxel albumin nanoparticles. INJ: Commercially available injection; HSA NP: Docetaxel albumin nanoparticles;

[0054] Figure 4 The relationship between tumor volume and time in human non-small cell lung cancer A549 after administration of low-dose (DNPL), high-dose (DNPH), DTX injection and blank group of the docetaxel albumin nanocomposition of the present invention is shown.

[0055] Figure 5 The changes in body weight over time in normal mice after administration of the docetaxel albumin nanocomposite or docetaxel injection according to the present invention are shown.

[0056] Figure 6 The relationship between the in vitro cytotoxicity of the DTX nanocomposite group and the solution in human non-small cell lung cancer A549 cells and time was shown.

[0057] Figure 7 The images show the appearance of the lyophilized cake (left) and the liquid (right) after reconstitution of the docetaxel albumin nanocomposition of the present invention. Detailed Implementation

[0058] The purpose of this invention is to overcome the defects and redundancy of existing formulations, while also addressing the poor stability of current docetaxel albumin nanoparticles. It fills the gap in the monitoring of docetaxel-related substances, protein dimers, and lysophospholipids, providing a stable docetaxel albumin nanocomposition with a formulation and preparation process suitable for industrial-scale production. This invention prepares poorly soluble docetaxel into an injectable tumor-targeting albumin nanocomposition, which, compared to existing formulations, exhibits higher drug loading, better tumor targeting effect and sustained-release effect, and the preparation process is simple and suitable for large-scale industrial production.

[0059] Docetaxel-Albumin Nanocomposite

[0060] The soluble albumin nanocomposition for injection described in this invention disperses and stabilizes docetaxel and a stabilizer after binding with albumin. This invention utilizes the high binding rate of docetaxel to albumin, allowing the two to naturally form albumin nanoparticles. The stabilizer acts as a solubilizer and dispersant for poorly soluble drugs, ultimately yielding a soluble albumin nanocomposition for injection.

[0061] The essential difference between the formulation of this invention and the prior art lies in the fact that the stabilizer is used as a medium to disperse and stabilize the drug and albumin complex, together with the drug and albumin to form a stable nano-formulation, wherein the phospholipid does not form liposomes on its own.

[0062] Specifically, the injectable docetaxel-albumin nanocomposition of the present invention is characterized by being made of docetaxel, albumin, stabilizer, and pharmaceutically necessary excipients, wherein the mass ratios of docetaxel, albumin, and stabilizer are 1-30%, 5-50%, and 5-85%, respectively, and the total of all components is 100%. Preferably, the mass ratios of docetaxel, albumin, and phospholipid are 1.6-7.2%, 10-40%, and 50-80%, respectively.

[0063] In this invention, the preferred albumin nanocomposition comprises: 1-3 wt% docetaxel, 15-25 wt% albumin, 70-85 wt% stabilizer; and optional lyophilized pharmaceutical excipients; or the formulation comprises: 5-10 wt% docetaxel, 30-40 wt% albumin, 50-70 wt% stabilizer; and optional lyophilized pharmaceutical excipients. In a more preferred embodiment, the albumin nanocomposition comprises: 1.5-2 wt% docetaxel, 18-22 wt% albumin, 75-80 wt% stabilizer; and optional lyophilized pharmaceutical excipients; or the formulation comprises: 6-8 wt% docetaxel, 30-35 wt% albumin, 55-65 wt% stabilizer; and optional lyophilized pharmaceutical excipients.

[0064] In this invention, the albumin is human serum albumin or bovine serum albumin, but human serum albumin is preferred.

[0065] In this invention, the phospholipids are not particularly limited and can be selected from (but are not limited to): egg yolk lecithin (EPC), soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), dilauroyl lecithin (DLPC), distearyl 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), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), dipalmitoyl phosphatidylethanolamine (DPPA), and dipalmitoyl phosphatidylethanolamine (DPPA). It is derived from one or more of the following: sphingomyelin (DPSP), dipalmitoyl lecithin (DPPC), dipalmitoyl phosphatidyl diserine (DPPS), dilauroyl phosphatidylglycerol (DLPG), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidyl phosphatidyl acid (DMPA), dimyristoyl phosphatidyl ethanolamine (DMPE), cerebrophosphatidyl serine (PS), dimyristoyl lecithin (DMPC), dimyristoyl phosphatidyl serine (DMPS), brain sphingomyelin (BSP), distearyl phosphatidyl glycerol (DSPG), distearyl sphingomyelin (DSSP), or distearyl phosphatidyl ethanolamine (DSPE); preferably EPC, SPC, DSPE, or HSPC.

[0066] The docetaxel albumin nanocomposite prepared by this invention is stable for more than 24 hours at room temperature and more than 72 hours at 4°C, without causing excessive increases in docetaxel-related substances, stabilizer-related substances, and protein dimers. Specifically, docetaxel-related substances increase by only 0.04%, and protein dimers increase by only 1.45%. The peroxide value, free fatty acids, LPE, and LPC of stabilizers such as phospholipids are all within the pharmacopoeia requirements, which is beneficial for industrial-scale production. The resulting lyophilized powder injection has a good appearance, rapid reconstitution, and its properties after reconstitution are very similar to those of the liquid before lyophilization. It remains stable for 24 hours at both room temperature and 4°C, greatly facilitating clinical application.

[0067] Preparation of docetaxel-albumin nanocomposite

[0068] This invention employs an emulsification method to prepare injectable albumin nanocomposites, with the particle size further controlled by selective ultrasonic or high-pressure homogenization processes. In one embodiment of this invention, docetaxel in a predetermined formulation is dissolved in an appropriate amount of ethanol or other suitable solvent, and phospholipids, fatty acids, and other stabilizers are dissolved in an appropriate amount of dichloromethane. The mixture of these two is used as the oil phase. Albumin is dissolved in an appropriate amount of deionized water (aqueous phase). The oil phase is then dropped into the magnetically stirred aqueous phase at room temperature or in an ice-water bath, and stirring is continued for a certain period of time. Then, ethanol and dichloromethane are removed by rotary evaporation in a water bath at 40-60°C to obtain the albumin nanocomposites loaded with docetaxel.

[0069] In this invention, after the docetaxel drug is prepared into albumin nanoparticles, the dispersion medium can be water, 5% glucose solution or physiological saline.

[0070] Docetaxel-Albumin Nanocomposition Formulation

[0071] Due to its favorable pharmacokinetic properties, the soluble injectable albumin nanocomposition of the present invention can be used to prepare drugs for treating cancer, or as a drug for cancer treatment; wherein the cancers include lung cancer, breast cancer, ovarian cancer, glioma, liver cancer, pancreatic ductal carcinoma, esophageal cancer, gastric cancer, pancreatic cancer, thyroid cancer, nasopharyngeal carcinoma, 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 cancers, sarcomas, or carcinosarcomas originating from the gallbladder, oral cavity, peripheral nervous system, mucous membranes, glands, blood vessels, bone tissue, lymph nodes, and eyes.

[0072] In this invention, the soluble albumin nanocomposition for injection can be administered via intravenous, subcutaneous, intramuscular, intramembranous, intraperitoneal, or other routes.

[0073] In this invention, after the docetaxel drug is encapsulated, the dosage of docetaxel drug is 0.01 to 20 mg / kg each time, preferably 1 to 10 mg / kg each time; the dosing regimen is daily or intermittent dosing, and the dosage for each course of treatment is 0.3 to 600 mg / kg, preferably 4 to 40 mg / kg per course of treatment.

[0074] The soluble injectable albumin nanocomposition of the present invention has been shown in pharmacodynamic tests to have a better therapeutic effect than commercially available injectables.

[0075] This invention utilizes the enhanced penetration and retention effect (EPR effect) of nanoparticles on tumors, allowing more drugs to passively target and concentrate in tumor tissue, thereby improving its anti-tumor effect. Simultaneously, due to the high bioavailability of the injection method and the passive targeting effect of the described soluble injectable albumin nanocomposite, the dosage can be significantly reduced, effectively lowering drug concentrations at non-target sites and helping to reduce drug toxicity. The described soluble injectable albumin nanocomposite avoids the disadvantages of low bioavailability and poor patient compliance of commercially available oral formulations, combining the water-soluble enhancement effect of albumin nanoparticles with the passive targeting effect, showing promising clinical application prospects. Furthermore, the preparation method of this invention is simple, with high drug recovery rate, suitable for large-scale industrial production.

[0076] Compared with the prior art, the soluble injectable albumin nanocomposition of the present invention has the following significant advantages:

[0077] ① It has a significant solubilizing effect on docetaxel, and its solubility is sufficient for clinical injection applications;

[0078] ②The encapsulation rate is almost 100%, there is no loss during the preparation process, the recovery rate is higher than 85%, and the drug loading is also high;

[0079] ③ The soluble injectable albumin nanocomposite can utilize the EPR effect at the tumor site and the active targeting effect of SPARC-gp60 to increase the accumulation of drugs in tumor tissues, which is conducive to exerting the anti-tumor effect of the drugs and reducing the toxic side effects on other tissues.

[0080] ④ It has a sustained-release effect, which can continuously kill tumor cells and reduce the frequency of administration.

[0081] ⑤ The formulation is stable, remaining stable for more than 24 hours at room temperature and more than 72 hours at 4°C, and does not cause an excessive increase in docetaxel-related substances, lysophospholipids and protein dimers, which is beneficial for mass production.

[0082] ⑥ The prepared lyophilized powder injection has a good appearance, fast reconstitution speed, and its properties after reconstitution are very similar to those before lyophilization. It can still be stable for 24 hours at room temperature and 4℃, which greatly facilitates clinical application.

[0083] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0084] Example 1: Formulation and preparation process of docetaxel albumin nanocomposite

[0085] Table 1. Proportions (mg) of each component in the docetaxel-loaded albumin nanocomposite formulation.

[0086]

[0087]

[0088] Docetaxel from Formulation F01-20 in Table 1 was dissolved in an appropriate amount of chloroform or other suitable solvent, and the stabilizer was dissolved in an appropriate amount of ethanol. The two were mixed together to form the oil phase. Albumin was dissolved in an appropriate amount of deionized water (aqueous phase). The oil phase was dropped into the aqueous phase at room temperature or in an ice-water bath. The mixture was stirred and dispersed for a certain period of time for initial emulsification. Then, it was nano-dispersed in a high-pressure homogenizer or emulsifier. The organic solvent was then removed by rotary evaporation or thin-film evaporation in a 50°C water bath to obtain an albumin nanocomposition containing docetaxel.

[0089] The results are shown in Table 2. The average particle size of the obtained formulations varied with different components, ranging from 50 to 150 nm. The recoveries were all above 85%, and the encapsulation rates were all above 98%. Since the stability of the formulations prepared in this invention cannot be reflected by particle size, and in previous formulation screening processes, visible precipitation occurred without significant changes in particle size, we used the turbidity change rate to measure the stability of the formulations (see the notes in Table 2 for the specific calculation method). When drug precipitation occurred, the turbidity change rate was large, while when no drug precipitation occurred, the turbidity change rate fluctuated within a limited range, generally within 10%.

[0090] As shown in Table 2, the preparations made from the prescriptions listed in the table can be stable for more than 24 hours at room temperature, and some prescriptions can even be stable for 48 hours. Under 4℃ conditions, they can be stable for more than 72 hours.

[0091] Table 2 Results of formulations with different component ratios

[0092]

[0093]

[0094] Note: Turbidity change rate = (A T -A0) / A0*100%, where A0 represents the absorbance of the nano-suspension at 350nm at time T0, and A T This indicates the absorbance after being left for T hours.

[0095] Example 2: Characterization of docetaxel albumin nanocomposite - morphology and particle size

[0096] A trace volume of albumin nanocomposite was placed on a copper grid, and the morphology of the nanocomposite was observed using phosphotungstic acid negative staining. The particle size of the prepared docetaxel albumin nanocomposite was measured using a dynamic light scattering particle size analyzer, and the average particle size was found to be 132.4 ± 0.59 nm. The electron microscopy and particle size distribution results are shown below. Figure 1 As shown in the figure. The results show that the nanocomposition of the present invention is spherical, with a round and relatively uniform appearance.

[0097] Example 3: In vitro stability test

[0098] (1) Experimental methods

[0099] Dilute HSA (20%) to 5% with PBS, set the particle size analyzer temperature to 37°C, and dilute the drug with 5% HSA solution to drug concentrations of 1, 10, and 100 μg / ml. Measure the particle size at 0, 10, 20, 30, and 60 min after dilution.

[0100] (2) Experimental Results

[0101] Experimental results are as follows Figure 2 As shown, at low concentrations, the nanoparticles quickly dissociate to form a drug-albumin complex, which is more conducive to drug penetration at the tumor site.

[0102] Example 4 Drug Release Experiment

[0103] (1) Experimental methods

[0104] Docetaxel injection and docetaxel albumin nanocomposite were diluted to 1 mg / ml. 1 ml of each was added to a dialysis bag with a molecular weight of 10000 kDa. The dialysis bags were placed in PBS at pH 5.0 and pH 7.4, respectively, with 3 samples per group. The samples were placed on a shaker (37℃, 100 rpm) and 1 ml was taken at 0.1, 0.25, 0.5, 1, 2, 4, 8, 16, 2, 48, and 72 h for analysis. After each sampling, 1 ml of fresh release medium was added.

[0105] (2) Experimental Results

[0106] Experimental results are as follows Figure 3 As shown, the injection precipitated after 2 hours, thus failing to produce a burst release effect. The release of the injection was not significantly different at the two pH levels. Albumin nanoparticles were released more completely under acidic conditions, reaching 91%.

[0107] Example 5: Pharmacodynamic study of docetaxel-loaded albumin nanocomposition against subcutaneous non-small cell lung cancer

[0108] Tumor cells: Human non-small cell lung cancer A549 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum.

[0109] Animals: Balb / c nude mice, 4 weeks old, male. Six nude mice were used in each of the experimental and negative control groups. Tumor cell inoculation: A subcutaneous inoculation model was used in the right axilla. Under aseptic conditions, vigorously growing A549 cells were harvested, digested, centrifuged, and resuspended to a cell suspension of approximately 2.0 E7 / ml. 0.1 ml of the cancer cell suspension was then subcutaneously inoculated into the right axilla of each mouse.

[0110] The calculation methods for tumor volume and tumor inhibition rate are the same as before. Each group was administered the drugs for two weeks according to the dosing regimen in Table 3. The tumor inhibition effect is as follows: Figures 4-6 As shown in the figure. The results show that,

[0111] Table 3. Pharmacodynamics of Docetaxel Albumin Nanocomposition in Subcutaneous Non-Small Cell Lung Cancer Model Mice: Tumor Suppression Experiment Dosing Regimen

[0112] DTX injection IV, 10 mg / kg, once every three days DNP low dose IV, 5 mg / kg, once every three days High dose of DNP IV, 10 mg / kg, once every three days control group IV, 0.9% NaCl

[0113] Table 4 shows the pharmacokinetic parameters of the docetaxel albumin nanocomposite and the marketed solution in rats after administration.

[0114] Table 4. Pharmacokinetic parameters of docetaxel injection (commercially available) and docetaxel albumin nanocomposite.

[0115]

[0116]

[0117] The results showed that the docetaxel nanoformulation of the present invention can effectively improve pharmacokinetics after injection and increase the in vivo utilization of the drug.

[0118] Example 6: Docetaxel albumin nanocomposite with lyophilized support

[0119] The docetaxel-loaded albumin nanocomposite solution was freeze-dried with a lyophilization protectant (trehalose, sucrose, or mannitol), and its properties are shown in the following photograph. Figure 7 As shown, the surface of the freeze-dried powder is smooth and delicate, uniformly white and cake-shaped, full, without obvious collapse or cracks; it reconstitutes rapidly, the reconstituted solution is semi-transparent, and the particle size is <150nm and uniformly distributed.

[0120] Example 7: Solvent Residue

[0121] 1. Experimental Methods

[0122] (1) Weigh the lyophilized powder, then reconstitute the lyophilized powder and bring the volume to 10ml.

[0123] (2) Take 2 ml to 20 ml headspace vials, and then detect the contents of ethanol and dichloromethane using a gas chromatograph to calculate the residual solvent ratio.

[0124] 2. Experimental Results

[0125] The results of solvent residue in the freeze-dried products are shown in Table 7. As can be seen from the table, the ethanol and dichloromethane in the final product both meet the pharmacopoeia requirements (ethanol not higher than 0.5%; dichloromethane not higher than 0.06%).

[0126] Table 5 Solvent Residue in Freeze-Dried Products

[0127]

[0128] Example 8: Moisture Determination

[0129] 1. Experimental Methods

[0130] Weigh the freeze-dried powder, then place it 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.

[0131] 2. Experimental Results

[0132] The moisture content of the freeze-dried products is shown in Table 6, and the moisture content of the freeze-dried products meets the quality requirements.

[0133] Table 6 Moisture content of freeze-dried products

[0134]

[0135] Example 9: Determination of formulation impurities

[0136] 1. Experimental Methods

[0137] (1) Redissolve the lyophilized powder and bring the volume to 10ml.

[0138] (2) Take 1 ml of the reconstituted preparation into a 10 ml volumetric flask, add 2 ml of ethanol and 2 ml of acetonitrile, sonicate for 5 min, and then dilute to 10 ml with diluent (water: acetonitrile: acetic acid = 50: 50: 0.5).

[0139] (3) Centrifuge at 12000 rpm for 20 min, and take the supernatant for detection in a liquid chromatograph.

[0140] 2. Experimental Results

[0141] The results for related substances of docetaxel are shown in Table 7. The related substances of the prepared formulation increased by only 0.04%.

[0142] Table 7. Proportions of Docetaxel-related substances

[0143]

[0144] Example 10: Protein Dimer

[0145] 1. Experimental Methods

[0146] (1) Redissolve the lyophilized powder and bring the volume to 10ml.

[0147] (2) Take 1 ml of the reconstituted preparation into a 10 ml volumetric flask, add ultrapure water to make up to 10 ml, mix well and then detect it in a liquid chromatograph.

[0148] 2. Experimental Results

[0149] The protein dimer results are shown in Table 8. The related substances in the prepared formulation increased by only 1.45%.

[0150] Table 8. Results of determination of protein dimer content in docetaxel albumin nanocomposite

[0151]

[0152] Example 11: Phospholipid-related detection

[0153] 1. Experimental Methods

[0154] The test was performed according to the method for egg yolk lecithin (for injection) in Part IV of the 2015 edition of the Chinese Pharmacopoeia.

[0155] 2. Experimental Results

[0156] The phospholipid-related test results of the docetaxel albumin nanocomposite are shown in Table 9. As can be seen from the table, the contents of lipid peroxides, free fatty acids, LPC and LPE all meet the national pharmacopoeia standards.

[0157] Table 9 Phospholipid-related detections in docetaxel albumin nanocomposites

[0158]

[0159]

[0160] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A docetaxel albumin nanoformulation, characterized in that, The formulation comprises the following components: Docetaxel 1-3 parts by weight, albumin 15-25 parts by weight, stabilizer 70-85 parts by weight; and optional pharmaceutical lyophilized excipients; Alternatively, the formulation may consist of the following components: 5-10 parts by weight of docetaxel, 30-40 parts by weight of albumin, 50-70 parts by weight of stabilizer; and pharmaceutical lyophilized excipients; and the formulation may not contain antioxidants; the stabilizer may be selected from phospholipids; Furthermore, in the aforementioned formulation, docetaxel, albumin, and stabilizer together form an albumin nano-formulation; The albumin nano-formulation method includes the following steps: (1) Provide docetaxel, albumin and stabilizer, and pharmaceutical lyophilized excipients according to the formulation; and provide organic solvent and dispersion medium; (2) The docetaxel and stabilizer are dispersed in an organic solvent to form an oil phase, and albumin is dissolved in an appropriate amount of deionized water to form an aqueous phase; (3) The aqueous phase is placed in a high-pressure homogenizer or emulsifier for high-shear stirring. At -10 to 40°C, the oil phase is injected into the aqueous phase for emulsification while being stirred under high shear, thereby forming an emulsion. (4) The emulsion is subjected to high-pressure homogenization to obtain a nanoparticle emulsion; (5) The organic solvent is removed by depressurized rotation or thin-film evaporation of the emulsion to obtain a docetaxel-albumin nanocomposition.

2. The nano-formulation as described in claim 1, characterized in that, The albumin is selected from the group consisting of recombinant albumin, bovine serum albumin, human serum albumin, or combinations thereof.

3. The nano-formulation as described in claim 1, characterized in that, The albumin mentioned is human serum albumin.

4. The nano-formulation as described in claim 1, characterized in that, The pharmaceutical freeze-dried excipients are selected from the following group: sucrose, trehalose, lactose, glucose, mannitol, sorbitol, or combinations thereof.

5. The nano-formulation as described in claim 1, characterized in that, The proportion of the pharmaceutical freeze-dried excipients is 1-20% (m / m), based on the total mass of the preparation.

6. The nano-formulation as described in claim 1, characterized in that, The formulation also includes organic solvents and / or dispersion media.

7. The nano-formulation as described in claim 6, characterized in that, 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, 5% glucose solution, physiological saline, or combinations thereof.

8. The nano-formulation as described in claim 1, characterized in that, The formulation comprises the following components: 1.5-2 parts by weight of docetaxel, 18-22 parts by weight of albumin, 75-80 parts by weight of stabilizer; and pharmaceutical lyophilized excipients; or the formulation comprises the following components: 6-8 parts by weight of docetaxel, 30-35 parts by weight of albumin, 55-65 parts by weight of stabilizer; and pharmaceutical lyophilized excipients.

9. The method for preparing nano-formulations as described in claim 1, characterized in that, The method includes the following steps: (1) Provide docetaxel, albumin and stabilizer according to the formulation, as well as optional pharmaceutical lyophilized excipients, organic solvents and dispersion media; (2) The docetaxel and stabilizer are dispersed in an organic solvent to form an oil phase, and albumin is dissolved in an appropriate amount of deionized water to form an aqueous phase; (3) The aqueous phase is placed in a high-pressure homogenizer or emulsifier for high-shear stirring. At -10 to 40°C, the oil phase is injected into the aqueous phase for emulsification while being stirred under high shear, thereby forming an emulsion. (4) The emulsion is subjected to high-pressure homogenization to obtain a nanoparticle emulsion; (5) The organic solvent is removed by depressurized rotation or thin-film evaporation of the emulsion to obtain a docetaxel-albumin nanocomposition.

10. The method as described in claim 9, characterized in that, The high-shear stirring speed in step (3) is 500–10000 rpm; and / or The emulsification time in step (3) is 2–30 min; and / or In the high-pressure homogenization process, the pressure is 5000–30000 psi.

11. The method as described in claim 9, characterized in that, The method further includes: lyophilizing the obtained docetaxel-albumin nanocomposite.

12. The method as described in claim 9, characterized in that, The freeze-drying process includes the following steps: The docetaxel-albumin nanocomposite was mixed with pharmaceutical lyophilized excipients and then pre-frozen at -50 to -30°C for 4 hours. Sublimation was performed once at 0.01-0.03 mbar; The product is then subjected to a second sublimation at 20–30°C to obtain a freeze-dried product.

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