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

Docetaxel albumin nano composition and preparation method thereof Download PDF

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Publication number
CN114533682A
CN114533682A CN202011248335.7A CN202011248335A CN114533682A CN 114533682 A CN114533682 A CN 114533682A CN 202011248335 A CN202011248335 A CN 202011248335A CN 114533682 A CN114533682 A CN 114533682A
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albumin
docetaxel
parts
stabilizer
nano
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张奇志
程云龙
吴红兵
王峰
吴静
曹锦旭
王刘畅
庞晓莹
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Shanghai Xinhengji Pharmaceutical Technology Co ltd
Fudan University
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Shanghai Xinhengji Pharmaceutical Technology Co ltd
Fudan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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

Docetaxel albumin nano composition and preparation method thereof
Technical Field
The invention belongs to the field of pharmaceutical preparation, and relates to a composition or a compound used in the field of anti-tumor, in particular to a preparation method of a nano composition containing docetaxel, albumin and a stabilizer as a prescription.
Background
In recent years, the incidence of tumors has increased dramatically, and the treatment difficulty and mortality rate are high, which become the second leading cause of human death. About 1810 ten new cancer cases and 960 ten cancer death cases are reported in 2018 all over the world, wherein the death cases of Chinese malignant tumors exceed 233.8 ten thousands, the death rate is over 30 percent, and the Chinese accounts for 21 cancer patients in every 100 new cancer patients all over the world, so that the method becomes a serious social problem. At present, the development of the disease is delayed by the combined treatment of radiotherapy and chemotherapy in clinic for treating the advanced tumor, but the advanced tumor is accompanied by serious immune system inhibition or toxic and side effects, so that the survival rate and the life quality of a patient are difficult to effectively improve. Therefore, the search for a treatment means or a medicament with good curative effect and low toxic and side effect is always a research hotspot and an effort direction in the field of tumor treatment.
Docetaxel (docetaxel) is a new generation of taxane drug developed in recent years, the action mechanism of docetaxel is similar to that of paclitaxel, but the antitumor activity of docetaxel is 1.3-12 times that of paclitaxel, and docetaxel has higher efficiency in metastatic breast cancer and non-small cell lung cancer treatment. Although docetaxel has a better antitumor effect, therapeutic practice shows that it still has the following defects: (1) docetaxel is almost insoluble in water, a large amount of surfactant is required to be added into clinically used injections, such as commercially available docetaxel (Taxotere) containing high-concentration tween 80, including alcohol-free preparations approved by Eagle pharmaceutical companies by FDA in 2015, although the docetaxel can be stored at room temperature and has longer stability time than Taxotere after dilution, serious sensitization side effects in clinical application caused by a large amount of tween-80 contained in a prescription are still not solved, such as bone marrow suppression, anaphylactic reaction, neurotoxicity, skin erythema, cardiovascular adverse reactions and the like; (2) docetaxel is a cytotoxic drug and has no targeting property, the drug is rapidly distributed and metabolized systemically after the injection is administered to generate systemic toxic and side effects, and no antidote is available once a cancer patient is overdosed after long-term administration; (3) the drug delivery systems of fat emulsion, microemulsion, liposome and the like for docetaxel do not have great progress in the aspect of reducing adverse reactions and toxic and side effects.
In conclusion, a docetaxel pharmaceutical preparation with good curative effect and low toxic and side effects is not available in the field.
Disclosure of Invention
The invention aims to provide a docetaxel medicinal preparation with good curative effect and lower toxic and side effects.
In a first aspect of the present invention, there is provided a docetaxel protein nano-formulation, including:
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.
In another preferred embodiment, the average particle size of the nano preparation is 50-150 nm.
In another preferred embodiment, the preparation is freeze-dried powder; preferably, the average particle size of the lyophilized powder after reconstitution is 50-150 nm.
In another preferred embodiment, the preparation is an injection.
In another preferred embodiment, the injection further comprises 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 embodiment, the docetaxel albumin nano composition comprises 1.6-7.2% of docetaxel albumin, 10-40% of albumin, 50-80% of stabilizer, and the total amount of all the components is 100% by total weight of the docetaxel albumin, the albumin and the stabilizer.
In another preferred embodiment, the 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.
In another preferred embodiment, the human serum albumin contains fatty acids.
In another preferred embodiment, the stabilizing agent is selected from the group consisting of phospholipids, fatty acids, amino acids, or combinations thereof; phospholipids are preferred.
In another preferred embodiment, 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 phosphatidylethanolamine, dipalmitoyl glycerol, dipalmitoyl phosphatidic acid, dipalmitoyl sphingomyelin, dipalmitoyl lecithin, dipalmitoyl phosphatidyldilserine, dilauroyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dimyristoyl phosphatidic acid, dimyristoyl phosphatidylethanolamine, cephalyl serine, dimyristoyl lecithin, dimyristoyl phosphatidylserine, cephalyl sphingomyelin, distearoyl phosphatidylcholine, with, Distearoyl phosphatidyl glycerol, distearoyl sphingomyelin, or distearoyl phosphatidyl ethanolamine, or a combination thereof.
In another preferred embodiment, the phospholipid does not readily form liposomes in the formulation.
In another preferred embodiment, the fatty acid is selected from the group consisting of: medium chain oil, medium long chain oil, palm kernel oil, coconut oil, squalene, or a combination thereof.
In another preferred embodiment, the compound is selected from the group consisting of: at least one of arginine, cysteine, lysine and proline.
In another preferred embodiment, the pharmaceutical lyophilization excipient is 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.
In another preferred example, when the lyophilization auxiliary material is selected from sucrose, trehalose, lactose and glucose, the proportion of the lyophilization auxiliary material is 5-20% (m/m).
In another preferred example, when the lyophilization auxiliary material is selected from mannitol and sorbitol, the proportion of the lyophilization auxiliary material is 1-10% (m/m).
In another preferred embodiment, the preparation further comprises 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; the dispersion medium is selected from the group consisting of: water, a 5% glucose solution, physiological saline, or a combination thereof.
In another preferred embodiment, the organic solvent is a mixed solvent of ethyl acetate and ethanol, wherein the proportion of ethanol is 10-50% (v/v).
In another preferred embodiment, the formulation comprises: 1-3 parts of docetaxel, 15-25 parts of albumin and 70-85 parts of a stabilizer; and optionally a pharmaceutically acceptable 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.
In another preferred embodiment, the preparation comprises: 1.5-2 parts of docetaxel, 18-22 parts of albumin and 75-80 parts of stabilizer; and optionally a pharmaceutically acceptable 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 pharmaceutically acceptable lyophilization excipient.
In a second aspect of the present invention, there is provided a method of preparing a nano-formulation according to the first aspect of the present invention, the method 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 nanoparticle 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.
In another preferred embodiment, the method is an emulsion method.
In another preferred embodiment, 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 another preferred example, the step (2) is performed at a temperature of 0 ℃ to 25 ℃.
In another preferred example, the step (3) is performed at a temperature of 0 ℃ to 25 ℃.
In another preferred example, the high pressure homogenization is performed at a temperature of 0 ℃ to 15 ℃.
In another preferred example, in the step (5), the reduced-pressure rotation or the thin-film evaporation is performed at a temperature of 25 ℃ to 60 ℃.
In another preferred example, in the step (5), the reduced pressure rotation or the thin film evaporation rotation speed is completed in a range of 20rpm to about 400 rpm.
In another preferred example, in the step (5), the reduced pressure rotation is performed under a vacuum degree of 0.1MPa to about 0.06 MPa.
In another preferred example, the method further comprises: and carrying out ultrasonic or high-pressure homogenization treatment on the docetaxel-albumin nano composition to obtain the nano composition with uniform particle size.
In the high-pressure homogenizing process, the pressure is 5000-30000 psi.
In another preferred example, in the high-pressure homogenizing process, the homogenizing time is 5-60 min.
In another preferred example, 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 at 0.01-0.03mbar (preferably, the sublimation comprises maintaining at-35-25 deg.C for 48h, and then maintaining at-10-0 deg.C for 12 h);
carrying out secondary sublimation (preferably 6 hours) at 20-30 ℃ to obtain a freeze-dried product.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a transmission electron microscope image of the prepared docetaxel albumin nano-composition and a distribution graph of particle size at room temperature for 24 h;
figure 2 stability of docetaxel albumin nanoparticles in 5% HSA mock plasma. A.1. mu.g/ml; b.10 mu g/ml; c.100 mu g/ml;
fig. 3 shows the release of docetaxel injection and docetaxel albumin nanoparticle drug. INJ.: commercially available injections; HSA NP: docetaxel albumin nanoparticles;
FIG. 4 shows the relationship between the volume of human non-small cell lung cancer A549 tumor bodies and time after the administration of the docetaxel albumin nano-composition of the present invention in low Dose (DNPL), high Dose (DNPH) formulations, DTX injection and blank group;
figure 5 shows the change in body weight over time following administration of docetaxel albumin nano-composition or docetaxel injection solution to normal mice in the present invention;
FIG. 6 shows the cytotoxicity of DTX nanocomposite set and human non-small cell lung cancer A549 in vitro solution versus time in accordance with the present invention;
figure 7 shows the appearance of lyophilized cake before reconstitution and liquid after reconstitution (right) of docetaxel albumin nanocomposite lyophilized formulation of the present invention.
Detailed Description
The invention aims to overcome the defects and prescription redundancy of the existing preparation, simultaneously overcome the poor stability of the existing docetaxel albumin nanoparticles, fill the blank that related substances, protein dimers and lysophospholipids of docetaxel are not monitored, provide a stable docetaxel albumin nano composition, and industrially amplify the prescription and the preparation process. The tumor targeting albumin nano composition for injection prepared from the insoluble docetaxel medicine has higher medicine-loading rate, good tumor targeting effect and sustained-release effect compared with the existing preparation, and the preparation process is simple and convenient, so that the preparation method is suitable for industrial mass production.
Docetaxel-albumin nano composition
According to the soluble albumin nano composition for injection, the docetaxel medicine and the stabilizer are dispersed and stabilized after being combined by albumin. The invention utilizes the characteristic of high combination rate of the docetaxel medicine and the albumin to enable the docetaxel medicine and the albumin to be naturally formed and prepared into albumin nanoparticles, and the stabilizer plays a role in solubilizing and dispersing the insoluble medicine to finally prepare the soluble albumin nano composition for injection.
The essential difference between the preparation of the present invention and the prior art is that the stabilizing agent is used as a medium for dispersing and stabilizing the complex of the drug and albumin, and the stabilizing agent, the drug and albumin together form a stable nano preparation, wherein the phospholipid does not form liposome alone.
Specifically, the docetaxel drug albumin nano composition for injection is characterized by being prepared from docetaxel drugs, albumin, a stabilizer and pharmaceutically necessary auxiliary materials, wherein the docetaxel drugs, the albumin and the stabilizer respectively account for 1-30%, 5-50% and 5-85% in mass ratio, and the sum of the components is 100%. Preferably, the docetaxel medicament, the albumin and the phospholipid respectively account for 1.6-7.2%, 10-40% and 50-80% in mass ratio.
In the present invention, a preferred albumin nanocomposite comprises: 1-3 wt% of docetaxel, 15-25 wt% of albumin and 70-85 wt% of stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient; or the formulation comprises: 5-10 wt% of docetaxel, 30-40 wt% of albumin and 50-70 wt% of stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient. In a more preferred embodiment, the albumin nanocomposite comprises: 1.5-2 wt% of docetaxel, 18-22 wt% of albumin and 75-80 wt% of stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient; or the formulation comprises: 6-8 wt% of docetaxel, 30-35 wt% of albumin and 55-65 wt% of stabilizer; and optionally a pharmaceutically acceptable lyophilization excipient.
In the present invention, the albumin is human serum albumin or bovine serum albumin, but human serum albumin is preferred.
In the present invention, the phospholipid is not particularly limited, and may 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 phosphatidylethanolamine (DPPE), dipalmitoyl glycerol (DPPG), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl sphingomyelin (DPSP), dipalmitoyl lecithin (DPPC), dipalmitoyl phosphatidyldilserine (DPPS), dilauroyl phosphatidylglycerol (DLPG), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), One or more of dimyristoyl phosphatidylethanolamine (DMPE), brain Phosphatidylserine (PS), dimyristoyl lecithin (DMPC), dimyristoyl phosphatidylserine (DMPS), Brain Sphingomyelin (BSP), distearoyl phosphatidylglycerol (DSPG), distearoyl sphingomyelin (DSSP), or distearoyl phosphatidylethanolamine (DSPE); EPC, SPC, DSPE or HSPC are preferred.
The docetaxel albumin nano composition prepared by the invention can be stabilized for more than 24 hours at room temperature and can be stabilized for more than 72 hours at 4 ℃, and excessive increase of related substances of docetaxel, related substances of a stabilizer and protein dimers can not be caused, wherein the related substances of docetaxel are only increased by 0.04%, the related substances of protein dimers are only increased by 1.45%, and the peroxide value, free fatty acid, LPE and LPC of the stabilizer such as phospholipid are in the pharmacopoeia requirement range, thereby being beneficial to industrial amplification production. The freeze-dried powder injection prepared by the method has good appearance and high redissolution speed, has small difference between the characters after redissolution and the liquid before freeze-drying, can be still stable for 24 hours at room temperature and 4 ℃, and is greatly convenient for clinical application.
Preparation of docetaxel-albumin nano composition
The invention adopts an emulsification method to prepare the albumin nano composition for injection, and can selectively adopt an ultrasonic or high-pressure homogenization process to further control the particle size. In one embodiment of the invention, docetaxel in a given prescription is dissolved in a proper amount of ethanol or other proper solvents, stabilizers such as phospholipid, fatty acid and the like are dissolved in a proper amount of dichloromethane, and the two are mixed to be used as an oil phase; dissolving albumin in a proper amount of deionized water (water phase), dripping the oil phase into the water phase stirred by magnetic force at room temperature or in ice water bath, continuously stirring for a certain time, and then carrying out rotary evaporation in water bath at 40-60 ℃ to remove ethanol and dichloromethane to prepare the docetaxel drug-loaded albumin nano composition.
In the invention, after the docetaxel medicine is prepared into the albumin nanoparticles, the dispersion medium can be water, 5% glucose solution or normal saline.
Docetaxel-albumin nano composition preparation
Due to good pharmacokinetics, the soluble albumin nano composition for injection can be used for preparing a medicament for treating cancer or used as a medicament for treating cancer; wherein the cancer comprises lung cancer, breast cancer, ovarian cancer, brain glioma, liver cancer, pancreatic duct cancer, esophageal cancer, stomach 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, primary or secondary cancer, sarcoma or carcinosarcoma and the like originating from gall bladder, oral cavity, peripheral nervous system, mucosa, gland, blood vessel, bone tissue, lymph node, eye.
In the invention, the soluble albumin nano composition for injection can be injected and administered by intravenous, subcutaneous, intramuscular, intramembranous, intraperitoneal or other routes.
In the invention, after the docetaxel medicine is encapsulated, the administration dose of the docetaxel medicine is 0.01-20 mg/kg per time, and the preferable administration dose is 1-10 mg/kg per time; the administration scheme is daily administration or interval administration, the administration dosage of each course is 0.3-600 mg/kg, and the preferred administration dosage of each course is 4-40 mg/kg.
Pharmacodynamic tests show that the albumin nano composition for soluble injection has better treatment effect than the commercial injection.
According to the invention, the enhanced permeation and retention effects (EPR effect) of the nanoparticles on the tumor are utilized, so that more drugs are passively targeted and concentrated in tumor tissues, and the anti-tumor effect of the drugs is improved; meanwhile, due to the high bioavailability of the injection mode and the passive targeting effect of the soluble albumin nano composition for injection, the administration dosage can be greatly reduced, so that the concentration of the medicine at a non-target part is effectively reduced, and the toxic and side effects of the medicine are reduced; the albumin nano composition for soluble injection overcomes the defects of low bioavailability and poor patient compliance of oral preparations on the market, fuses the water solubility improving effect and the passive targeting effect of the albumin nano particles, and has good clinical application prospect. On the other hand, the preparation method is simple, has high drug recovery rate and is suitable for industrial mass production.
Compared with the prior art, the soluble albumin nano composition for injection has the following remarkable advantages:
the docetaxel injection has obvious solubilization effect on docetaxel, and the solubility of the docetaxel injection is sufficient for clinical injection application;
secondly, the encapsulation efficiency is almost 100 percent, the preparation process has no loss, the recovery rate is higher than 85 percent, and the drug-loading rate is also higher;
and thirdly, the soluble albumin nano composition for injection can increase the accumulation of the medicament in tumor tissues by utilizing the EPR effect of tumor parts and the active targeting effect of SPARC-gp60, is favorable for playing the anti-tumor effect of the medicament and reduces the toxic and side effects on other tissues.
Has slow release function, can continuously kill tumor cells and reduce the administration frequency.
The preparation is stable, can be stable for more than 24 hours at room temperature and can be stable for more than 72 hours at 4 ℃, does not cause excessive increase of related substances of docetaxel, lysophospholipid and protein dimer, and is beneficial to mass production;
the freeze-dried powder injection prepared by the method has good appearance and high redissolution speed, has small difference between the characters after redissolution and before freeze-drying, can be still stable for 24 hours at room temperature and 4 ℃, and is greatly convenient for clinical application.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1: prescription and preparation process of docetaxel albumin nano composition
TABLE 1 proportions (mg) of the components of the docetaxel-loaded albumin nanocomposite formulations
Figure BDA0002770787240000091
Figure BDA0002770787240000101
Respectively dissolving docetaxel in formulas F01-20 in table 1 in proper amount of chloroform or other proper solvents, dissolving stabilizer in proper amount of ethanol, and mixing the two to obtain oil phase; dissolving albumin in a proper amount of deionized water (water phase), dripping an oil phase into the water phase at room temperature or in an ice water bath, stirring and dispersing colostrum for a certain time, performing nano dispersion in a high-pressure homogenizer or a milk homogenizer, and removing an organic solvent by water bath rotary evaporation or thin film evaporation at 50 ℃ to obtain the albumin nano composition containing docetaxel.
The results obtained are shown in Table 2. The average particle size of the preparation varies with different components, and the recovery rate is above 85% and the encapsulation rate is above 98% in 50-150 nm. Because the stability of the preparation prepared by the invention cannot be embodied by the particle size, the preparation can be separated out by naked eyes in the screening process of the previous prescription, but the particle size does not change obviously, and therefore, the turbidity change rate is adopted to balance the stability of the preparation (the specific calculation mode is shown in table 2). When the drug is precipitated, the turbidity change rate is large, and when no drug is precipitated, the turbidity change rate varies within a limited range, basically within 10%.
As shown in Table 2, the formulations prepared by the in-table recipe can be stable for more than 24 hours at room temperature, some formulations can be stable for even 48 hours, and the formulations can be stable for more than 72 hours at 4 ℃.
Table 2 results of different ratio component formulations
Figure BDA0002770787240000102
Figure BDA0002770787240000111
Note: turbidity change rate ═ aT-A0)/A0*100％,A0Denotes the absorbance at 350nm of the nanosuspension at T0, ATThe absorbance of the resulting solution was measured after standing for T hours.
Example 2: characterization of docetaxel albumin nanocomposites-morphology and particle size
Taking a micro-volume albumin nano composition on a copper mesh, adopting a phosphotungstic acid negative dyeing method, and observing the form of the nano composition by a transmission electron microscope. The particle size of the prepared docetaxel albumin nano-composition is measured by a dynamic light scattering particle sizer, the average particle size is 132.4 +/-0.59 nm, and the electron microscope and the particle size distribution result are respectively shown in figure 1. The results show that the nano-composition of the invention is spherical, round in appearance and relatively uniform.
Example 3: in vitro stability assay
(1) Experimental method
HSA (20%) was diluted to 5% with PBS, the temperature of the particle sizer was adjusted to 37 ℃, the drug was diluted with a 5% HSA solution to drug concentrations of 1, 10, 100. mu.g/ml, and the particle size was measured at 0, 10, 20, 30, 60min after dilution.
(2) Results of the experiment
The experimental result is shown in fig. 2, under the condition of low concentration, the nanoparticles are quickly dissociated to form a compound of the drug and albumin, so that the penetration of the drug at the tumor part is more facilitated.
Example 4 drug Release test
(1) Experimental methods
Diluting docetaxel injection and docetaxel albumin nano composition to 1mg/ml, adding dialysis bags with molecular weight of 10000kDa into 1ml respectively, placing the dialysis bags in PBS with pH value of 5.0 and pH value of 7.4, 3 parts each, placing in a shaking table (37 ℃, 100rpm), taking 1ml in 0.1, 0.25, 0.5, 1, 2, 4, 8, 16, 2, 48 and 72h, detecting, and adding 1ml of fresh release medium after each sampling.
(2) Results of the experiment
The experimental results are shown in fig. 3, and the injection has drug precipitation after 2h, thus causing no burst effect of the drug. The release of the injection at the two pH values is not very different. The albumin nanoparticles are completely released under an acidic condition, and can reach 91 percent.
Example 5: pharmacodynamic test of docetaxel-loaded albumin nano composition on subcutaneous non-small cell lung cancer
Tumor cells: human non-small cell lung carcinoma a549 cells (purchased from shanghai cell bank of chinese academy of sciences) were cultured in DMEM medium containing 10% fetal bovine serum.
Animals: balb/c nude mice, 4 weeks old, male. Test and negative control groups 6 nude mice per group. Tumor cell inoculation: adopting a right axillary subcutaneous inoculation model, taking the vigorous A549 cells under aseptic condition, digesting the cells, centrifuging and suspending the cells to about 2.0E7/ml cell suspension, and inoculating 0.1ml cancer cell suspension under the right axillary subcutaneous of each mouse.
Tumor volume and tumor inhibition rate were calculated as before, and the tumor inhibition effect was shown in fig. 4 to 6 when each group was administered for two weeks according to the administration schedule of table 3. The results show that the content of the active ingredients,
TABLE 3 pharmacodynamic anti-tumor experimental dosing regimen of docetaxel albumin nanocomposite on subcutaneous non-small cell lung cancer model mice
DTX injection i.v.,10mg/kg, once in three days
DNP Low dose i.v.,5mg/kg, once in three days
High DNP dose i.v.,10mg/kg, once in three days
Control group i.v.,0.9％Nacl
Table 4 shows the pharmacokinetic parameters of the docetaxel albumin nanocomposite of the present invention and the marketed solution after administration to rats.
TABLE 4 pharmacokinetic parameters of docetaxel injection (commercially available) and docetaxel albumin nanocomposite
Figure BDA0002770787240000121
Figure BDA0002770787240000131
The result shows that the docetaxel nanometer preparation can effectively improve the pharmacokinetics after injection and increase the in vivo utilization rate of the medicine.
Example 6: docetaxel albumin-entrapped nano composition and freeze-drying supporting agent
The docetaxel carrying albumin nano composition solution is added with a freeze-drying protective agent (trehalose, sucrose or mannitol) for freeze drying, the character photo of the freeze-dried powder is shown in figure 7, the surface of the freeze-dried powder is smooth and fine, uniform white cake-shaped, full and free of obvious collapse and cracks; the re-dissolution is rapid, the re-dissolution solution is semitransparent, and the particle size is less than 150nm and is uniformly distributed.
Example 7: solvent residue
1. Experimental methods
(1) Weighing the freeze-dried powder, then redissolving the freeze-dried powder, and fixing the volume to 10 ml.
(2) And (3) taking a 2ml to 20ml headspace bottle, detecting the contents of ethanol and dichloromethane by a gas chromatograph, and calculating the residual ratio of the solvent.
2. Results of the experiment
The solvent residue results of the lyophilized products are shown in Table 7, and it can be seen from the table that ethanol and dichloromethane of the final products both meet the pharmacopoeia requirements (ethanol is not higher than 0.5%; dichloromethane is not higher than 0.06%).
TABLE 5 Freeze-dried product solvent residue
Figure BDA0002770787240000132
Example 8: moisture determination
1. Experimental methods
Weighing the weight of the freeze-dried powder, then placing the freeze-dried powder in a titration bottle of a moisture tester, inputting the mass of the freeze-dried powder, and automatically calculating the moisture contained in the freeze-dried powder by the moisture tester.
2. Results of the experiment
The water content of the freeze-dried product is shown in table 6, and the water content of the freeze-dried product meets the quality specification.
TABLE 6 Freeze-dried product moisture
Figure BDA0002770787240000141
Example 9: determination of impurities in formulations
1. Experimental methods
(1) Redissolving the freeze-dried powder and fixing the volume to 10 ml.
(2) Taking 1ml of the redissolved preparation to a 10ml volumetric flask, adding 2ml of ethanol and 2ml of acetonitrile, carrying out ultrasonic treatment for 5min, and then carrying out constant volume to 10ml by using a diluent (water: acetonitrile: acetic acid: 50: 0.5).
(3) Centrifuging at 12000rpm for 20min, and detecting the supernatant with liquid chromatograph.
2. Results of the experiment
Docetaxel-related substance results are shown in table 7, and the preparation of the completed formulation-related substance increased only 0.04%.
TABLE 7 docetaxel relative material ratios
Figure BDA0002770787240000142
Example 10: protein dimers
1. Experimental methods
(1) Redissolving the freeze-dried powder and fixing the volume to 10 ml.
(2) Taking 1ml of the redissolved preparation to a 10ml volumetric flask, adding ultrapure water to the volumetric flask to reach a constant volume of 10ml, uniformly mixing, and detecting by a liquid chromatograph.
2. Results of the experiment
Protein dimer results are shown in table 8, with only a 1.45% increase in the preparation related substances.
TABLE 8 determination results of protein dimer content of docetaxel albumin nano-composition
Figure BDA0002770787240000151
Example 11: phospholipid-related assays
1. Experimental methods
The detection is carried out according to the method of yolk lecithin (for injection) in the fourth part of the year of pharmacopoeia 2015 of China.
2. Results of the experiment
The relevant detection results of phospholipid in the docetaxel albumin nano composition are shown in table 9, and the table shows that the contents of lipid peroxide, free fatty acid, LPC and LPE all accord with the national pharmacopoeia standard.
TABLE 9 phospholipid-related assays in docetaxel albumin nanocomposites
Figure BDA0002770787240000152
Figure BDA0002770787240000161
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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.
CN202011248335.7A 2020-11-10 2020-11-10 Docetaxel albumin nano composition and preparation method thereof Pending CN114533682A (en)

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