CN102327230B - A protein nanoparticle encapsulating taxane drugs and its preparation method - Google Patents

Abstract

The invention belongs to the field of pharmacy, and discloses a protein nanoparticle encapsulating taxane drugs and a preparation method thereof. The formulation of the granules contains the following substances in weight percentage: 0.1-10% of taxane drugs, 0.1-40% of water-soluble carrier materials, and 50-90% of protein substances. In the method, taxane drugs and water-soluble carrier materials are first prepared into a water-soluble carrier solid dispersion containing taxane drugs; then the obtained solid dispersion is added to an aqueous medium containing protein substances, mixed evenly, The mixture is processed under high shear conditions to obtain a suspension of protein nanoparticles encapsulated with insoluble drugs; and then further prepared into the desired dosage form. The protein nanoparticles have the advantages of high drug loading, uniform particle size, good stability, and high safety. This method avoids toxic organic solvent residues, improves the safety of clinical medication, and has simple process, low cost, and strong operability. .

Description

A protein nanoparticle encapsulating taxane drugs and its preparation method

technical field

The invention belongs to the field of pharmacy, and relates to a preparation method of protein nanoparticle encapsulating taxane drugs.

Background technique

Taxane drugs (such as paclitaxel, docetaxel, etc.) are one of the most effective anti-tumor drugs currently used clinically. Paclitaxel is a natural product isolated from the bark or needles of Taxus or its species discovered in the 1970s. Docetaxel is a semi-synthetic product, which was later discovered to be a class of anti-tumor agents with a special anti-tumor mechanism of action .

The anti-tumor mechanism of taxane drugs is to promote microtubule polymerization and reduce the depolymerization speed of microtubules, so that the microtubules are in a stable non-functional state, thereby achieving the purpose of preventing tumor cell mitosis and proliferation. And preclinical studies have shown that compared with paclitaxel, docetaxel has stronger affinity for microtubules, longer plasma half-life and longer intracellular residence time.

Although taxane drugs have good anti-tumor effect, their water solubility is low (about 10ug/ml), so paclitaxel injection and docetaxel injection used clinically need to use surfactant polymer respectively. Oxyethylene castor oil and polysorbate 80 (Tween-80) and co-solvent ethanol were used for the purpose of dissolving the paclitaxel drug. Paclitaxel injection and docetaxel injection were first successfully developed and marketed by Bristol-Myers Squibb of the United States and Sanofi-Aventis of France respectively.

Although it can be prepared into injection after using surfactant polyoxyethylene castor oil and polysorbate 80, its toxic and side effects in clinical application are relatively large, and it is easy to cause more complications, and the more common ones include allergic reactions, bone marrow Inhibition (mainly manifested as neutropenia), fluid retention, neurotoxicity, hair loss and other adverse reactions.

A large number of studies have shown that polyoxyethylene castor oil can promote the release of a large amount of histamine in the body, which can cause allergic reactions, and can also cause nerve conduction delay sensory neuropathy. In addition, polyoxyethylene castor oil can also form tiny particles in the blood to encapsulate paclitaxel molecules, which affects the diffusion of drug molecules to tissues, thereby affecting its anti-tumor effect. Studies have also shown that polyoxyethylene castor oil can dissolve Diethylenehexyl Phthalate (Diethylenehexy Phthalate) in PVC infusion sets. However, polysorbate 80 (Tween-80) is hemolytic and has a high viscosity, making it inconvenient for clinical use. The above-mentioned injection preparations have certain problems in terms of toxicity, side effects, administration convenience and stability, etc., so it is necessary to develop new dosage forms of taxanes.

A number of biocompatible polymers can be used to prepare polymeric shells for encapsulating water-insoluble pharmacologically active drugs. In fact, any polymer, natural or synthetic, can be used to form a layer around a practically water-insoluble pharmacologically active drug as long as it can contain sulfhydryl or disulfide bonds in its structure as required. Disulfide cross-linked shell. Proteins containing cysteine and/or disulfide bonds are pharmaceutically acceptable biological carriers, and this patent takes human serum albumin as an example.

Human serum albumin (human serum albumin, HSA) is a single-chain non-glycosylated protein composed of 585 amino acids, with a molecular weight of 66.5kDa. It is the most abundant protein in human plasma (accounting for 50%-60% of the total human plasma protein). %), has the characteristics of stable chemical performance, safety and non-toxicity, non-immunogenicity, good biocompatibility, biodegradability, and long circulation in the body (half-life up to 19 days).

As an anti-tumor drug carrier material, HSA has many advantages, mainly including:

①Excellent drug loading potential: HSA polypeptide chain has more polar groups and hydrophobic amino acids, and has a high affinity for 70% of small molecule drugs, especially insoluble drugs, and can undergo reversible binding. Its payload and drug release provide the possibility; the secondary structure of albumin contains about 48% of α-helical structure, 15% of β-pleated sheet structure, and the rest is random coil structure, so it has a lot of network gaps, which is Carrying drugs creates favorable spatial conditions.

② Broad-spectrum tumor targeting: albumin is an important nitrogen source for tumor cells, and fast-growing tumors have a strong ability to uptake and reserve albumin. HSA, as an anti-tumor drug carrier, binds to the albumin receptor on the vascular endothelial cell membrane, activates the kiln protein on the cell membrane, and transfers the anti-tumor drug to the tumor cell interstitium. Tumor cell interstitium and tumor cell surface are rich in a large number of cysteine acidic secretory proteins, which function similar to albumin receptors and can specifically attract and adhere to albumin, while cysteine acidic secretory proteins are in Almost all tumor tissues (bladder, liver, ovary, kidney, digestive tract, breast, etc.) are overexpressed, which forms a drug storage pool in tumor tissues, which is a broad spectrum of albumin nanocarriers loaded with antitumor drugs Targeted killing offers the possibility.

③Structure modifiability: The HSA structure contains a large number of reactive groups, such as free amino groups, carboxyl groups, sulfhydryl groups, etc., which can be coupled with anti-tumor drugs through chemical bonds to improve drug solubility, increase in vivo circulation time and tumor targeting ; It can also be coupled with "target molecules" such as antibodies or ligands to further improve the tumor targeting performance of HSA nanocarriers. In recent years, it has also been reported that HSA is coupled with polymer materials such as dextran, aldohexose, or polyisopropylacrylamide to improve the physical and chemical properties of HSA as a drug carrier material.

In view of the unique advantages of HSA as a drug carrier material, the current research on albumin-based drug delivery systems at home and abroad mainly focuses on two directions:

① Prodrugs: chemical coupling of drugs, especially antineoplastic drugs (doxorubicin, paclitaxel, docetaxel, methotrexate, mitomycin, 5-fluorouracil) by using a large number of reactive groups of HSA The free amino group, carboxyl group or sulfhydryl group of HSA can be used to improve drug solubility, improve circulation time in vivo and tumor targeting, and there have been many reports abroad.

However, the direct chemical coupling of HSA and drugs has the following problems: 1) The range of drug selection is narrow: the drug needs to have a certain reactive group, and the drug is prone to failure during the chemical coupling process; 2) The curative effect is uncertain Performance: The ease of breaking the chemical bond between the drug and HSA determines the efficacy of the drug. If it breaks too fast, HSA will lose its advantage as a carrier material. If it breaks too slowly, the drug will not be able to exert its curative effect; 3) High cost and low drug loading : In the coupling process, the drug dosage is far greater than that of HSA, and the amount of coupled drug is limited, resulting in large preparation costs (especially the high price of anti-tumor drug raw materials) and low drug loading in the system.

②Nano-delivery system: Physical encapsulation of drugs can significantly increase the loading of carrier materials on drugs. At present, the preparation or drug loading processes of albumin nanoparticles reported at home and abroad mainly include: emulsification coacervation method, desolvation method, pH coagulation method and Nab technology.

Although the first three methods have the longest research and application time and the most reports, there are still many problems:

1) The particle size is large. Nanoparticles have a particle size of 0.5-10 μm and are easily affected by operating conditions. There are few reports on albumin nanoparticles smaller than 200nm. Too large a particle size is not conducive to the accumulation of nanocarriers in tumors through the EPR effect of tumors;

2) Curing is required. Because HSA is highly water-soluble, the prepared nanoparticles need to be cured, that is, add a chemical cross-linking agent to cross-link albumin or heat to denature the protein. The former is more toxic, and it is easy to cause the drug to cross-link at the same time and lose its curative effect. , the latter is not suitable for the loading of temperature-sensitive drugs; in addition, some people think that the method of cross-linking or heat curing will reduce the hydrophilicity of the surface of HSA nanoparticles, thereby reducing the circulation time in the blood, which is not conducive to tumor targeting;

3) The post-processing process is complicated. Due to the use of toxic additives such as surfactants, oils, and chemical cross-linking agents in the process, a large amount of organic solvents are required to clean and purify, but a small amount of residue will still bring hidden dangers to the safety of medication; high-speed centrifugation (16000-20000g) is used to collect nano Particles, not only have high requirements for instruments, but also have poor redispersibility in water;

4) The range of applicable drugs is small. It is only suitable for the loading of water-soluble drugs, and a considerable part of anti-tumor drugs are poorly soluble drugs.

Nab technology is a drug-binding albumin nanotechnology established by American bioscience companies in recent years. This technology is only suitable for the encapsulation of fat-soluble drugs with a relatively high plasma protein binding rate. In 2005, paclitaxel-binding albumin nanoparticles prepared by this technology (Kai ) has been approved by the FDA for marketing, and is mainly used for metastatic breast cancer after failure of combined chemotherapy or breast cancer recurrence within 6 months of adjuvant chemotherapy.

)具有更低的毒性、更好的疗效，但是也存在不少缺陷，包括：1)毒性溶剂的使用。采用氯仿为溶媒，虽有真空蒸发操作，但增加了工艺难度，并且产品可能有溶剂残留；2)载药量低。仅能控制在2-10wt％，需消耗大量的HSA；3)体内外稳定性差。该制剂在pH＞5.8条件下，药物在几小时内极易析出，因此制剂pH值需控制在pH5.4-5.8范围内(在此范围也仅稳定24h)。而人体内环境pH值为7.4，该制剂进入血液循环以后，预计药物短时间将从载体析出而不利于肿瘤的特异性摄取，目前临床上用药剂量是泰

制剂的1.5倍。Compared with the traditional polyoxyethylene castor oil preparation (Thai

) has lower toxicity and better curative effect, but there are also many defects, including: 1) the use of toxic solvents. Adopting chloroform as solvent, though vacuum evaporation operation, increases process difficulty, and product may have solvent residue; 2) drug load is low. It can only be controlled at 2-10 wt%, and a large amount of HSA needs to be consumed; 3) the stability in vivo and in vitro is poor. Under the condition of pH > 5.8, the drug is easily precipitated within a few hours, so the pH value of the preparation should be controlled within the range of pH 5.4-5.8 (it is only stable for 24 hours in this range). The pH value of the human body environment is 7.4. After the preparation enters the blood circulation, it is expected that the drug will be precipitated from the carrier in a short time, which is not conducive to the specific uptake of the tumor. The current clinical dosage is Thailand

1.5 times that of the preparation.

Since preparation methods such as ultrasonic technology cannot be used for industrial scale production, and the particle size obtained by it is too large, this makes it unsuitable and cannot be used for drug administration to patients. Therefore, U.S. Biosciences Co., Ltd. has recorded and claimed in patents US2007082838 and CN98808225 and CN97199720 to protect the freeze-dried preparations of docetaxel/paclitaxel and human serum albumin that can be reconstituted by using a high-pressure homogenization method, and the resulting reconstituted The stability of the suspension exceeds 24 hours.

High-shear technology has super mixing, crushing, dispersing, emulsifying and other functions. It has a particularly remarkable crushing effect on polymer elastic materials such as SBS and SBR. It can effectively solve the problems of crushing and emulsifying various materials, replacing the traditional one. Colloid mill equipment. Compared with the traditional production process, it has the advantages of low energy consumption, low production cost, high product quality and ultra-fine.

In CN98808225 and CN97199720 patents, drug delivery systems containing water-insoluble drugs and protein-coated particles and their preparation methods are described and claimed, wherein the average diameter of the particles is 10-200nm. The preparation method of the drug combination system is to place the mixture of the organic phase containing the water-insoluble drug dispersed therein and the protein-containing aqueous medium in a high-pressure homogenizer of 3000-30000Psi, and subject it to high-shear Cut processing produces the particles described above, and the composition is free of surfactants.

The present inventor has repeatedly repeated the embodiments of the above-mentioned patent, particularly the embodiments 1, 5 and 6 of CN97199720, but never obtained the results described in the embodiments and claims of this patent. The inventors prepared the disclosed mixture and then treated the mixture with an Avestin high pressure homogenizer in its recommended pressure range to obtain a nanoemulsion with pH = 6.8 and evaporated with a rotary evaporator as described in the patent Removing the solvent can produce nanoparticles with an average particle size of about 220nm, which can be easily reconstituted into a suspension after adding physiological saline after freeze-drying, but the size of the nanoparticles increases significantly compared with that before freeze-drying, and the average particle size is determined by freezing The 220nm before drying increased to 390nm, and the precipitation of nanoparticles appeared within 4h. In addition, filtration is carried out with the microporous membrane described in the patent CN97199720, but the result is that the filter is easily blocked, and the drug yield is lower than 30%, which is different from the 70-100% yield result stated in the patent .

In the US2007082838 patent, it is recorded and claimed that the albumin nanoparticles containing docetaxel drug added with stabilizer sodium citrate and/or sodium chloride have an average particle size of <200nm, and as described in the patent, these are The nanoemulsion obtained by the method is said to have high stability, and the meaning of term "stability" here has not only expressed that the average particle size does not change with time or the freeze-drying process, but also indicates that the drug precipitation of nanoparticles does not occur (US200708283, implemented Example 12).

The present inventor has repeatedly repeated the embodiments of the above patents, especially the embodiments 11, 16, and 18 of US200708283, but has never obtained the results disclosed in the patent embodiments and claims. The inventors prepared the disclosed mixture, and then treated it with an Avestin high-pressure homogenizer within its recommended pressure range to obtain a nanoemulsion with pH = 7.2, and after removing the organic solvent by evaporation with a rotary evaporator, the average particle size Nanoparticles of about 280 nm. But the precipitation of nanoparticle occurs very soon, it is more difficult when carrying out described microporous membrane filtration (1.2um, 0.8um, 0.45um and 0.22um), and the situation of clogging occurs easily in filter membrane, and its freeze-dried product is in The nanosuspension formed after reconstitution in the physiological solution is unstable, and precipitation visible to the naked eye appears within about 8 hours, which is completely different from the result claimed in the patent that the stability is greater than 24 hours.

Contents of the invention

The purpose of the present invention is to address the above-mentioned problems and shortcomings of the prior art, and the present invention provides a protein nanoparticle with simpler preparation process, more stable properties and better clinical application properties.

Another object of the present invention is to provide a method for preparing the above-mentioned protein nanoparticles.

In the process of preparing protein nanoparticles, the inventors accidentally found that when water-soluble carrier materials (such as polyethylene glycol, mannitol, PVP, etc.) were added to the protein system, the protein loaded with taxanes Nanoparticles are easier to form, and at the same time have uniform particle size and improved stability. In order to investigate the effect of this water-soluble carrier material in the system, the inventor has studied the relationship between the water-soluble carrier material and proteins, surprisingly, the results of transmission electron microscopy and differential scanning calorimetry all reflect that, The two are not a simple mixed system, but form a new composite system through interaction. We speculate that due to the formation of this new composite system, the taxane drugs can be better entrapped, thereby significantly improving the operability of the preparation process and making the nanoparticles more stable and uniform.

Due to the significant advantages of this new composite system, the inventors then tried to mix the water-soluble carrier with the taxane drug first, so that the taxane drug exists in the water-soluble carrier in the form of molecular dispersion, and the two are in the form of solid The form of dispersion is added to the protein solution, mixed and subjected to high shear force to form protein nanoparticles of taxane drugs. It was found that protein nanoparticles are easier to form and have better stability; at the same time, the nanoparticles have obvious long-term blood circulation and tissue and organ targeting, and have better pharmacodynamic properties.

The invention provides a preparation method for adding taxane drugs (such as paclitaxel, docetaxel, etc.) to a nanoscale carrier in the form of molecular dispersion. This method is easier to obtain taxane drug nanoparticles (average diameter less than 200 nanometers) than nab technology, and does not need to add any toxic organic solvents (such as dichloromethane, chloroform, etc.), avoiding organic solvents in the preparation The residues in the drug greatly improve the safety of clinical medication.

At the same time, the present invention provides a protein nanoparticle, which has a high drug-loading capacity for taxane drugs, and can significantly reduce the amount of protein substances; the protein nanoparticle also has good in vivo and in vitro stability, and the liquid preparation It can be stored stably for a long time at room temperature, improving the convenience and safety of medication. The nano-particles of taxane drugs obtained by the method have obvious long-term blood circulation and tissue and organ targeting, and therefore have better pharmacodynamic properties. In summary, the present invention provides a protein nanoparticle with simpler preparation process, more stable properties and better clinical application properties. The protein nanoparticle has the characteristics of uniform particle size, good stability, good safety and the like.

The purpose of the present invention is achieved through the following technical solutions:

A protein nanoparticle encapsulating taxane drugs, the formula of the protein nanoparticle contains the following substances by weight percentage:

0.1-10% taxane drugs, 0.1-40% water-soluble carrier materials, 50-90% protein substances.

The protein nanoparticle, wherein the taxane drugs are selected from one or more of the following substances: paclitaxel, docetaxel, or their derivatives.

The protein nanoparticles, wherein the water-soluble carrier material is selected from one or more of the following substances: polyethylene glycol, polyvinylpyrrolidone, poloxamer, polyethylene oxide, mixed fatty acid esters, Mannitol, urea, sodium alginate, sodium hydroxypropyl cellulose, polyacrylic acid resin, gelatin, sodium carboxymethyl cellulose.

The proteinaceous substance referred to in the present invention refers to the following substances that can be cross-linked by thiol and/or disulfide bonds: naturally occurring or synthetic proteins and their derivatives, synthetic protein polymers and their derivatives, natural or synthetic proteinoids and their derivatives, or their mixtures.

Said naturally occurring proteins include albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme, alpha-2-macroglobulin, fibronectin, vitronectin, fibrinogen, lipase; said The synthetic protein polymer is selected from one or more of the following substances containing free sulfhydryl and/or disulfide modifications: polyethylene glycol, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycol, Polyglycolide, polycaprolactone or its copolymer, synthetic polyamino acid.

The preparation method of the protein nanoparticle, the method comprises the following steps:

a. making taxane drugs and water-soluble carrier materials into water-soluble carrier solid dispersions containing taxane drugs;

b. adding the obtained solid dispersion into an aqueous medium containing protein substances, mixing evenly, and processing the mixture under high shear conditions to obtain a suspension of protein nanoparticles coated with taxane drugs;

c. Dry the suspension or first pass through sterile filtration and then dry, and make a solid preparation, semi-solid preparation or gas preparation of protein nanoparticles coated with taxane drugs according to the required dosage form;

or

The suspension obtained in step b is directly used as a liquid preparation or further prepared into other types of liquid preparations, or the suspension is dried or sterile filtered and then dried before being reconstituted into a liquid preparation.

The preparation method, wherein the aqueous medium is selected from water for injection, pure water, mannitol solution, phosphate aqueous solution, dextran solution, glucose aqueous solution, sodium chloride aqueous solution, amino acid solution, vitamin solution, carbohydrate solution, or any of them A mixture of two or more.

The preparation method, wherein the method for preparing the water-soluble carrier solid dispersion containing taxanes is a melting method, a solvent method, a solvent-melting method, a solvent-spray (freeze) drying method, a grinding method, or a double helix method. Extrusion method; and these methods do not introduce any organic solvent or any other organic solvent except ethanol; preferably adopt solvent-melt method; (methods such as melting method, solvent method, solvent-melt method are the conventional methods for preparing solid dispersion Method. For example, the medicine is dissolved in ethanol (solvent), the water-soluble carrier is heated and melted (melted), and the two are mixed, which is the solvent-melt method. Another example is that the medicine is heated and melted, the water-soluble carrier is heated and melted, and the two are mixed , which is the melting method. If it is a solvent-melting method, it is "no need to introduce any other organic solvents except ethanol" that is, only ethanol is used. If it is a melting method, it is "no need to introduce any organic solvent" The solvent-melting method is preferably used in the present invention. Compared with the melting method, the advantage of the solvent-melting method is that some drugs are directly melted, which will lead to excessive temperature, degradation, and stability cannot be guaranteed. Therefore, this patent mainly uses solvent-melting The specific method can be determined according to the nature of the selected drug and carrier.)

The high shear condition refers to the application of equipment with high pressure and high shear force at ^the same time, so that the mixture can achieve efficient mixing, pulverization, dispersion and emulsification; wherein, high pressure refers to a pressure range of 5000 to 30000 psi, Preferably 6000-25000 ^psi .

Said preparation method, wherein the average particle size of the protein nanoparticles is 20-1000 nm, preferably 20-200 nm.

Said preparation method, wherein the drying method adopts freeze-drying or spray-drying; the sterile filtration adopts 0.22 μm filter to filter.

Specifically, the present invention provides a preparation method for adding taxane drugs (such as paclitaxel, docetaxel, etc.) to a nanoscale carrier in the form of molecular dispersion. This method is easier to obtain small nanoparticles of taxane drugs (average diameter less than 200 nanometers) than nab technology, and does not need to add any toxic organic solvents (eg, dichloromethane, chloroform, etc.). The nano-particles of taxane drugs obtained by the method are more stable, have high drug-loading capacity, have obvious long-term blood circulation and tissue and organ targeting, and therefore have better pharmacodynamic properties.

This method provides a method for forming nanoparticles of taxanes by desolvation techniques, for example, high shear forces (such as sonication, high-pressure homogenization or similar conditions) can be used in the absence of any conventional surfactants. and any polymeric core material that forms the matrix of the nanoparticles.

The method provides a method for the preparation of reusable small nanoparticles (less than 200 nm in diameter) that can be sterile filtered through a 0.22 micron filter. The preparation of nanoparticles of a size that can be filtered through a 0.22 micron filter is significant because formulations containing large amounts of any protein such as albumin cannot be sterilized by conventional methods such as autoclaving because heat can denature the protein . Of course, if a filter with a larger pore size is used, the average particle size of the particles can also be controlled at about 1000 nm, but in the present invention, the average particle size of the particles is preferably controlled at 20-200 nm.

Add protein substances (for example, human serum albumin) to the aqueous medium in a concentration range of about 0.05-25% (w/v, g/ml, the same below), and in the range of 0.5%-10% (w/v) Inside is better. Such aqueous media are physiological saline, buffered saline, water, buffered aqueous media, amino acid solutions, vitamin solutions, carbohydrate solutions or similar media, and mixtures of any two or more of these media. Unlike conventional nanoparticle formation methods, no surfactants (e.g., sodium lauryl sulfate, lecithin, Tween 80, polyol F68, and similar compounds, etc.) or toxic organic solvents (e.g., dichloromethane, chloroform, etc.).

Prepare taxanes (paclitaxel, docetaxel, etc.) and water-soluble carriers (such as polyethylene glycol, polyvinylpyrrolidone, poloxamer, etc.) A solid dispersion (powder) of a taxane drug and a water-soluble carrier. The formation of this solid dispersion does not need to introduce any other organic solvents except ethanol, even does not need to introduce organic solvents at all, avoiding the residue of organic solvents (such as chloroform, dichloromethane, etc.) in the preparation, greatly improving clinical drug use security.

Homogenization under low shear forms a mixture of micro and nano droplets. This can be accomplished by means known to those skilled in the art, for example, using a conventional laboratory homogenizer operating in the range of about 2,000 to about 15,000 rpm.

Drug-loaded nanoparticles are formed by homogenization under high shear conditions. This homogenization is usually carried out in a high pressure homogenizer, typically operating at a pressure in the range of 5,000 to 30,000 ^psig , preferably in the range of 6,000 to ^25,000 psig. The resulting emulsion contains extremely fine aqueous carrier nano-droplets (containing dissolved pharmacologically active substances, etc.) and extremely fine protein nano-droplets. Acceptable homogenization methods include devices that impart high shear and cavitation, such as high pressure homogenizers, ultrasonic processors, high shear mixers, and the like.

The liquid suspension can be dried to obtain the powder of protein nanoparticles encapsulating taxane drugs. The resulting powder can be redispersed at any suitable time and in a suitable aqueous medium to obtain a suspension which can be administered to mammals. Such aqueous media are physiological saline, buffered saline, water, buffered aqueous media, amino acid solutions, vitamin solutions, carbohydrate solutions or similar media, and mixtures of any two or more of these media. Methods employed to obtain such powders include freeze drying, spray drying, and similar techniques.

It can be further converted into a powder form by removing the contained moisture, for example, by vacuum freeze-drying in a suitable temperature-time range. The protein (e.g., human serum albumin) itself acts as a cryoprotectant without the use of conventional cryoprotectants such as mannitol, sucrose, glycerol, and similar compounds, and this powder can be Easily reassembled. Although not required, it is of course understood that such conventional cryoprotectants may be added to the formulations of the present invention if so desired.

According to one embodiment of the present invention, there is provided a method of forming extremely fine submicron particles (nanoparticles), ie, particles less than 200 nanometers in diameter. The particles can be sterile filtered prior to use in liquid suspension. It is important to be able to sterile filter the final product (i.e. drug granules) obtained during the formulation process of the present invention, because it is impossible to filter suspensions containing high concentrations of proteins (e.g., human serum albumin) using conventional methods such as autoclaves. Sterilize.

The application of the protein nanoparticle encapsulating taxane drugs in pharmacy, the protein nanoparticle can be used for gastrointestinal administration and parenteral administration. The preparation form of the protein nanoparticles can be solid dosage form, semi-solid dosage form, liquid dosage form and gas dosage form, including lyophilized powder, capsule, granule, ointment, paste, suspension, injection, spray, inhalation wait.

In the implementation of the present invention, many biocompatible protein substances can be used to prepare the carrier shell of protein nanoparticles, which can be wrapped on the outside of taxane drugs. In fact, any protein substance, natural or synthetic, can be used to form a layer of disulfide bonds around taxanes as long as it can contain sulfhydryl groups or disulfide bonds in its structure as required Cross-linked shell. Sulfhydryl or disulfide bonds may be pre-existing in the polymer structure or may be introduced by appropriate chemical modification, e.g. naturally occurring polymers such as proteins, peptides, polynucleotides, polysaccharides (e.g. starch, cellulose, dextran , algin, chitosan, pectin, hyaluronic acid, etc.), proteoglycans, lipoproteins, etc. are all candidates for this modification.

Proteins in the present invention include albumin (containing 35 cysteines), immunoglobulins, casein, lipoproteins, hemoglobin (each α ₂ β ₂ unit contains 6 cysteine residues), lysozyme Enzyme (containing 8 cysteine residues), immunoglobulin, α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, lipase, etc. Among them, proteins, peptides, enzymes, antibodies and their mixtures are protein substances commonly used in the present invention.

The presently preferred proteinaceous material for use in the present invention is albumin. For example, alpha-2-macroglobulin, a well-known opsonin, can be used to enhance the uptake of vector-encapsulated taxanes by macrophage-like cells, or can facilitate the administration of such vector-encapsulated taxanes. into the liver and spleen.

Likewise, synthetic polypeptides containing cysteine residues are good candidates for proteinaceous substances that form the outer coat of taxanes. In addition, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamide and polyvinylpyrrolidone, etc. are chemically modified (such as introducing sulfhydryl and/or disulfide bonds), and forming Good candidates for shells (eg, to cause cross-linking). Thus, examples of such materials contemplated for the practice of the present invention are synthetic polyamino acids containing cysteine residues and/or disulfide bonds; polyvinyl alcohols modified to contain free sulfhydryl groups and/or disulfide bonds; Polyhydroxyethyl methacrylate, modified to contain free thiols and/or disulfide bonds; polyacrylic acid, modified to contain free thiols and/or disulfide bonds; polyethoxazoline, modified to contain free thiols and and/or disulfide bonds; polyacrylamides, modified to contain free sulfhydryl groups and/or disulfide bonds; polyvinylpyrrolidone, modified to contain free sulfhydryl groups and/or disulfide bonds; polyglycols, modified to contain free sulfhydryl groups and/or disulfide bonds; polylactide, polyglycolide, polycaprolactone, or copolymers thereof, modified to contain free thiol groups and/or disulfide bonds; and any two or more of the foregoing mixture.

Beneficial effects of the present invention

The present invention provides a method for preparing protein nanoparticles encapsulating taxane drugs. In the method, suspended taxane drug particles are encapsulated in a biocompatible composite polymer shell with a diameter of to the nanoscale. The method does not need to introduce any other organic solvents except ethanol, even does not need to introduce organic solvents at all, avoids the residue of organic solvents (such as chloroform, dichloromethane, etc.) or emulsifiers, greatly improving the safety of clinical medication; this method also does not require the use of conventional surfactants. The method provides a more easily formed and more stable taxane drug nanoparticle, and the method has the advantages of simple process, low cost and strong operability. The method The present invention also provides a method for preparing nano particles capable of sterile filtration.

At the same time, the present invention provides a protein nanoparticle, the protein nanoparticle has a high drug loading capacity for taxanes, and can significantly reduce the amount of protein substances; the protein nanoparticle also has good in vivo and in vitro stability, liquid The preparation can be stored stably for a long time at room temperature, improving the convenience and safety of medication. The nano-particles of taxane drugs obtained by the method have obvious long-term blood circulation and tissue and organ targeting, and therefore have better pharmacodynamic properties. In summary, the present invention provides a protein nanoparticle with simpler preparation process, more stable properties and better clinical application properties. The protein nanoparticle has the characteristics of high drug loading, uniform particle size, good stability, high safety and the like.

Description of drawings

Fig. 1 is the strength particle size distribution diagram of paclitaxel albumin nano-preparation prepared by melting method. (The ordinate is the intensity percentage (%), and the abscissa is the particle size (nm), and Fig. 2, Fig. 7-Fig. 11 are all similar.)

Fig. 2 is the strength particle size distribution diagram of paclitaxel albumin nano-preparation prepared by melt-solvent volatilization method.

Figure 3 is a WAXD graph. Among them: sample a: paclitaxel powder, sample b: freeze-dried human serum albumin powder, sample c: physical mixture of paclitaxel and albumin, sample d: paclitaxel preparation sample.

Figure 4 is a DSC diagram. Among them: sample a: freeze-dried human serum albumin powder, sample b: paclitaxel powder; sample c: physical mixture of paclitaxel and albumin; sample d: paclitaxel preparation sample.

Figure 5 is a transmission electron microscope image. Wherein: sample a: human serum albumin freeze-dried powder, sample b: polyethylene glycol albumin powder, sample c: paclitaxel albumin nano preparation freeze-dried powder.

Fig. 6 is a fluorescence scan diagram. In the figure, along the direction of the curve a→h, the concentration of polyethylene glycol increases.

Fig. 7 is a distribution diagram of strength and particle size of docetaxel albumin nano-preparation.

Detailed ways

The present invention is described further below by embodiment.

Example 1 Preparation of Paclitaxel Albumin Nanoparticles by Melting Method

Dissolve 500 mg of paclitaxel in 9.0 mL of ethanol. After 1200 mg of polyethylene glycol was heated and completely melted in an oil bath at 60°C, the paclitaxel solution was added thereto, and stirred by magnetic force until it was completely mixed evenly. After the ethanol was removed by rotary evaporation, it was rapidly cooled under vigorous stirring conditions. After vacuum drying overnight, the solid dispersion was added to 85 ml of human serum albumin aqueous solution (4.5% w/v, g/ml, the same below). The mixture was premixed by a high-speed disperser (XHF-1, Shanghai Jinda Biochemical Instrument Factory) for 1 minute to form a coarse milk, and then transferred to a high-pressure homogenizer (EmulsiFlex-05, Avestin, Canada). The high-pressure homogenization is carried out under the condition of 5000-30,000 ^psi , and the emulsion is repeatedly circulated at least 5 times to obtain a protein nanoparticle suspension, and the obtained product has relatively dense opalescence. As measured by a laser particle size analyzer (Nano-ZS90, Malvern, UK), the obtained paclitaxel albumin nanoparticles generally have a diameter of 130-220 nanometers, and the results are shown in FIG. 1 .

The protein nanoparticle suspension was further lyophilized for 48 hours without adding any cryoprotectant, and the obtained powder could be easily reconstituted into a protein nanoparticle suspension by adding sterile water or saline. The size of the particles after reconstitution is about the same as before lyophilization.

Example 2 Preparation of paclitaxel albumin nanoparticles by melting-solvent evaporation method

Dissolve 300 mg of paclitaxel in 6.0 mL of ethanol. Dissolve 900 mg of polyethylene glycol in 1.5 ml of absolute ethanol, heat and completely melt in an oil bath at 45°C, add the paclitaxel solution into it, stir magnetically until it is completely mixed, and remove the ethanol by rotary evaporation, quickly under vigorous stirring conditions cool down. After drying under vacuum overnight, the solid dispersion was added to 65 ml of aqueous human serum albumin (4.5% w/v). The mixture was premixed by a high-speed disperser (XHF-1, Shanghai Jinda Biochemical Instrument Factory) for 1 minute to form a coarse milk, and then transferred to a high-pressure homogenizer (EmulsiFlex-05, Avestin, Canada). The emulsification is carried out under the condition of 5000-30,000 ^psi , and the emulsion is repeatedly circulated at least 6 times to obtain a suspension of protein nanoparticles. British Malvern Company) determined that the paclitaxel albumin nanoparticles obtained as a result generally have a diameter of 120-200 nanometers, and the results are shown in FIG. 2 .

The protein nanoparticle suspension was further lyophilized for 48 hours without adding any cryoprotectant, and the obtained powder could be easily reconstituted into a protein nanoparticle suspension by adding sterile water or saline. The size of the particles after reconstitution is about the same as before lyophilization.

Example 3 Preparation of Paclitaxel Albumin Nanoparticles Sterile Filterable Less than 200 Nanometers

Dissolve 500 mg of paclitaxel in 9.0 mL of ethanol. Dissolve 800 mg of polyethylene glycol in 1.5 ml of absolute ethanol, heat and completely melt in an oil bath at 45°C, add paclitaxel solution into it, stir magnetically until it is completely mixed evenly, and remove ethanol by rotary evaporation, rapidly under vigorous stirring conditions cool down. After drying under vacuum overnight, the solid dispersion was added to 97 ml of aqueous human serum albumin (4.5% w/v). The mixture was premixed by a high-speed disperser (XHF-1, Shanghai Jinda Biochemical Instrument Factory) for 1 minute to form a coarse milk, and then transferred to a high-pressure homogenizer (EmulsiFlex-05, Avestin, Canada). The emulsification is carried out under the condition of 10000-40,000 ^psi , and the emulsion is repeatedly circulated at least 6 times to obtain a suspension of protein nanoparticles. British Malvern Company) determined that the paclitaxel albumin nanoparticles obtained as a result generally have a diameter of 120-170 nanometers.

The protein nanoparticle suspension was filtered through a 0.22 micron millipore filter without any change in turbidity or particle size. The HPLC analysis of the paclitaxel content showed that more than 95% of the paclitaxel could be recovered after filtration, and the results are shown in Table 1. This method provides a sterile paclitaxel-albumin nanosuspension.

The sterile suspension was further lyophilized for 48 hours without adding any cryoprotectant, and the obtained powder could be easily reconstituted into protein nanoparticle suspension by adding sterile water or saline. The size of the particles after reconstitution is about the same as before lyophilization.

Table 1

Example 4 Determination of the Physical State of Paclitaxel in Nanoparticle Form by X-ray Powder Diffraction

X-ray powder diffraction can be used to determine the crystalline or non-crystalline nature of paclitaxel in lyophilized powder formulations. According to the X-ray diffraction (X-ray diffraction) principle, the peak shape of the diffraction peak is mainly related to the incompleteness of the crystal and the crystal structure, that is, the size of the grain size, defects and distortions in the crystal, etc. According to the Scherrer Equation, the grain size is inversely proportional to the square root of the diffraction peak, and the grain size reflects its crystallinity, that is, the wider the diffraction peak corresponds to the lower the crystallinity.

Take by weighing a small amount of sample a-paclitaxel powder; Sample b-freeze-dried human serum albumin powder; The physical mixture of sample c-paclitaxel and albumin; ), XD-3A powder diffractometer for powder X-ray diffraction test. The powder X-ray diffraction test was carried out under the conditions of a diffraction angle of 5-40°, a scanning speed of 1°/min, an operating voltage of 40kV, and an operating current of 50mA. The results are shown in Figure 3.

Sample a shows that the sample has three strong diffraction peaks at 5.28°, 8.84° and 12.36°, and several small diffraction peaks between 15° and 25°. Sample b shows the broad band bumps typical of amorphous materials. Sample c shows the broad-band ridges typical of amorphous materials, but in addition characteristic peaks at 5.28°, 8.84° and 12.36° are visible. The paclitaxel formulation of sample d did not show evidence of paclitaxel's crystallization properties, but was very similar to the broad-band bulge of amorphous material in sample b, suggesting that paclitaxel actually existed in a molecular state or an amorphous state in the nano-formulation, thus indicating that The drug paclitaxel was indeed present in the albumin nanocarriers after high pressure homogenization.

The DSC spectrum of embodiment 5 paclitaxel albumin nanoparticles

Differential Scanning Calorimetry (DSC) is a technique to measure the relationship between the power difference input to the sample and the reference sample and the temperature under programmed temperature control. Crystals absorb energy (J) to destroy the crystal lattice when melting. The amount of absorbed energy is related to the crystal structure. The characteristic constant enthalpy (ΔH, unit J·g ^-1 ) of a specific crystal form of a molecule can characterize its crystal form characteristics.

Weigh sample a-freeze-dried human serum albumin powder; sample b-paclitaxel powder; sample c-paclitaxel and albumin physical mixture; and sample d-paclitaxel albumin nanoparticle freeze-dried powder (prepared according to the embodiments of the present invention) 2~3mg each, heated by NETZSCH-DSC 204 analyzer at a heating rate of 10°C/min, the temperature range is 50~300°C, and the DSC analysis is carried out in a nitrogen atmosphere. The result is shown in Figure 4.

It can be seen from the figure that curve a shows that albumin has a small dehydration exothermic peak at 67.4°C and a slow melting peak at around 222.3°C; in curve b, the endothermic peak at 224.2°C and the exothermic peak at 244.5°C are the melting peaks of paclitaxel respectively and degradation peaks, these two characteristic peaks exist in the physical mixture of paclitaxel and albumin (curve c), but not in the paclitaxel-albumin nano-formulation (curve d). The above spectral data indicate that the drug exists in the micellar framework in the form of amorphous or solid solution.

Embodiment 6 Morphological study of paclitaxel albumin nanoparticles

Weigh an appropriate amount of sample a-human serum albumin freeze-dried powder, sample b-polyethylene glycol albumin powder and sample c-paclitaxel albumin nanoparticle freeze-dried powder (prepared according to the embodiment of the present invention), and mix them with 5% glucose solution Fully hydrated to obtain a light blue opalescent micellar solution, stained with phosphotungstic acid negative staining, that is, take 1 drop of the tested micellar solution and drop it in the groove of the spot reaction porcelain plate, and put the carbon-sprayed copper grid on the test plate. After 1-2 minutes, take out the copper mesh, and use a small piece of filter paper to absorb the residual liquid from the edge of the copper mesh; place the copper mesh on the dye solution (4% phosphotungstic acid solution, pH 7.0) for about 30 seconds, and dry the excess The dye solution was dried, and the morphology was observed with a Hitachi H-7000 transmission electron microscope. The results are shown in Figure 5.

As shown in the figure, human serum albumin cannot form a spherical structure, but is dispersed into a uniform solution state; after adding polyethylene glycol, human serum albumin shows a more regular spherical structure with a particle size of about 100nm; while the drug-loaded The albumin preparation forms a more regular and dense spherical structure with a particle size of about 120nm and a uniform particle size distribution. At the same time, it can be seen that the particle size results of sample b and sample c are smaller than the particle size obtained by dynamic light scattering experiment (respectively 122nm and 138nm), which may be due to the collapse of the micelle surface caused by the drying process in the TEM sample preparation process due to.

Embodiment 7 Fluorescence spectrometry research on the interaction between polyethylene glycol and human serum albumin

Human serum albumin contains tryptophan residues, and tryptophan can produce strong fluorescence when excited by ultraviolet light. From the results of fluorescence scanning, it can be seen that the best excitation wavelength of fluorescence of human serum albumin is 285nm, and the maximum emission wavelength is about 350nm.

Many small molecule drugs or water-soluble carriers can interact with albumin, causing fluorescence quenching. According to this principle, we can do a series of experiments to prove the interaction between other substances and albumin.

Add 1.0mL of 1.0×10 ^-5 mol·L ^-1 human serum albumin solution and different volumes of 1.0×10 ^-4 mol·L ^-1 polyethylene glycol solution to a 10ml colorimetric tube, and dilute with water to the scale, shake well, incubate at constant temperature in a constant temperature water bath for 10 min, fix Kex=285nm, Kem=350nm, measure relative fluorescence intensity^F=F ₀ -F (F, F ₀ represent the presence of polyethylene glycol and no polyethylene glycol respectively System solution fluorescence intensity when ethylene glycol exists), the result is as shown in Figure 6 (IF is the unit of fluorescence intensity, subscript F is the meaning of fluorescent fluorescence).

According to the fluorescence results, it can be seen that polyethylene glycol has a quenching effect on the endogenous fluorescence of human serum albumin under the condition of 25°C. As the concentration of polyethylene glycol increases (curve a→h), the fluorescence emission wavelength of albumin shifts blue, and the fluorescence intensity decreases simultaneously. The results showed that the interaction between polyethylene glycol and albumin was dominated by van der Waals force, which led to regular fluorescence quenching of albumin, and the quenching mechanism was consistent with dynamic quenching.

The effect of embodiment 8 drug concentration on paclitaxel albumin nanoparticles particle size

By changing the concentration of paclitaxel and keeping other parameters the same as those described in Example 2, a series of paclitaxel albumin nano-preparations were prepared. As measured by laser particle sizer, it was found that lower drug concentrations produced particles with a diameter of approximately 160 nm, while higher concentrations produced smaller particles with a diameter of approximately 120 nm. When the concentration of paclitaxel in the human serum albumin solution is 1 mg/ml, the particle diameter is about 250 nanometers; when the concentration of paclitaxel in the human serum albumin solution is 2 mg/ml, the particle diameter is about 180 nanometers; When the concentration of paclitaxel in human serum albumin solution is 5 mg/ml, the particle diameter is about 130 nanometers.

The compatibility stability of embodiment 9 commonly used transfusions

The compatibility stability of paclitaxel albumin nano freeze-dried preparation (prepared according to the embodiment of the present invention) and commonly used infusion solutions (eg, 5% glucose solution, 0.9% physiological saline) was investigated, so as to provide reference for clinical use. Generally speaking, the dilution stability only needs to reach 8 hours to meet clinical application. Therefore, in this example, the particle size and paclitaxel content were used as indicators to evaluate the compatibility stability within 8 hours.

The experimental results showed that paclitaxel albumin nano-lyophilized preparations could be reconstituted with 5% glucose or 0.9% normal saline to obtain a homogeneous opalescent solution, and as shown in Table 2, there was no significant change in each index within 8 hours. Therefore, both 5% glucose and 0.9% normal saline can be used as the reconstitution medium of paclitaxel albumin nanoparticle lyophilized preparation in clinical use.

Table 2

Particle size and potential changes of paclitaxel albumin nanoparticles before and after freeze-drying in embodiment 10

Zetasizer 3000HS laser particle size analyzer measured paclitaxel albumin nano freeze-dried powder (prepared according to the embodiment of the present invention) after reconstitution, compared with before freeze-drying, the average particle size increased slightly, and the polydispersity index did not change, indicating that paclitaxel albumin The basic properties of the nanoparticles do not change before and after freeze-drying, and the stability of the paclitaxel albumin nano-preparation is guaranteed through the solidification process of the freeze-drying.

In addition, the zeta potential remained basically unchanged before and after freeze-drying, both between -32 and -36mV. Generally speaking, when the absolute value of the Zeta potential is >30mV, due to the large amount of charge on the surface of the nanoparticles, there is a large repulsive force between the nanoparticles, which is conducive to the stability of the nano-solution and the uniform size of the nanoparticles. The absolute values of the Zeta potentials of the paclitaxel albumin nanoparticles were all >30mV, which indicated that both had good physical stability. The specific results are shown in Table 3.

table 3

Stability of Paclitaxel Albumin Nano-Preparation after Reconstitution of Example 11

The stability of paclitaxel albumin nano-lyophilized preparations after reconstitution in 0.9% normal saline was investigated to provide reference for clinical use, and the compatibility stability within 8 hours was evaluated by particle size and drug concentration.

The freeze-dried paclitaxel albumin nano-preparation (prepared according to the embodiment of the present invention) in the vial was redissolved with physiological saline to concentrations of 1, 2, and 5 mg/ml respectively, and stored at room temperature. The particle size and drug content were measured at different time points, and the suspension was stable for at least 7 days. The specific particle size and drug content of the 5 mg/ml formulation are shown in Table 4.

Table 4

Example 12 Mouse Tissue Distribution Experiment of Paclitaxel Albumin Nano-Preparation

(美国百时美施贵宝公司)，凯(美国生物科学公司)和紫杉醇白蛋白纳米制剂(按本发明实施例制备)，给药后分别于5、15min和0.5、1、3、8、12、24h时间点取一组(n＝6)，分别眼球取血后，颈椎脱臼处死，取心、肝、脾、肺、肾、脑、肿瘤等脏器。全血置于肝素钠试管中，离心，分离血浆；脏器用生理盐水冲洗，滤纸吸干其水分，称定总量(血液按小鼠体重的8％计)。提取出生物样品中的药物后进样，记录药物峰面积，计算血浆及各组织不同时间的药物浓度。根据药物浓度随时间变化曲线计算AUC、MRT及靶向效率。不同时间药物浓度如下表所示，其中表5为泰各组织中药物经时浓度，表6为凯各组织中药物经时浓度，表7为紫杉醇白蛋白纳米制剂各组织中药物经时浓度。Select 450 healthy Kunming female mice with a body weight of 20±2g, fast for 12h, divide them into 75 groups randomly, and inject Thai

(Bristol-Myers Squibb, USA), Kay (U.S. Biosciences Corporation) and paclitaxel albumin nano-preparation (prepared according to the embodiments of the present invention), after administration, take a group (n=6) at 5,15min and 0.5,1,3,8,12,24h time points respectively ), after taking blood from the eyeballs, they were killed by cervical dislocation, and their hearts, livers, spleens, lungs, kidneys, brains, tumors and other organs were harvested. The whole blood was placed in a sodium heparin test tube, centrifuged, and the plasma was separated; the organs were washed with normal saline, and the water was blotted with filter paper, and the total amount was weighed (the blood was calculated as 8% of the mouse's body weight). After extracting the drug in the biological sample, inject the sample, record the drug peak area, and calculate the drug concentration in plasma and various tissues at different times. AUC, MRT and targeting efficiency were calculated according to the curve of drug concentration versus time. The drug concentrations at different times are shown in the table below, where Table 5 is the Thai Time-dependent concentration of drugs in each tissue, Table 6 shows Drug concentration in each tissue over time, Table 7 shows drug concentration in each tissue of paclitaxel albumin nano-preparation.

各组织中药物经时浓度(ng/ml)Table 5 Thai

Drug concentration in each tissue over time (ng/ml)

Table 6 Kay Drug concentration in each tissue over time (ng/ml)

Table 7 Time-dependent drug concentration (ng/ml) in each tissue of paclitaxel albumin nano-preparation

From the results, it can be known that the time-dependent concentration of the drug in each tissue

凯

和紫杉醇白蛋白纳米制剂的AUC值分别为38.45、75.72、236.52；MRT分别为11.75、17.42、26.41；1) Thai

Kay

The AUC values of paclitaxel and albumin nano-preparations were 38.45, 75.72, 236.52; the MRT were 11.75, 17.42, 26.41;

凯

和紫杉醇白蛋白纳米制剂的肿瘤相对摄取率Re分别为1、1.969、6.151；显示出了本制剂相对于凯素更强的肿瘤靶向性。2) Thai

Kay

The tumor relative uptake rates Re of paclitaxel and albumin nano-preparations were 1, 1.969, and 6.151, respectively; it shows that this preparation has stronger tumor targeting than Kexol.

凯

和紫杉醇白蛋白纳米制剂的肿瘤靶向效率Te分别为0.88、5.97、10.76；显示出了本制剂更好的肿瘤靶向性。3) Thai

Kay

The tumor targeting efficiencies Te of paclitaxel and albumin nano-preparations were 0.88, 5.97, and 10.76, respectively; showing better tumor targeting of this preparation.

Rat pharmacokinetic experiment of embodiment 13 paclitaxel albumin nanoparticles

凯和紫杉醇白蛋白纳米制剂(按本发明实施例制备)。以7mg/kg的剂量由尾静脉注射给药，分别于给药后5，15，30min，1，2，4，6，8，12，24，36h眼眶取血约0.5mL，置于加有肝素钠的离心管内，离心10min(6000r/min)分离血浆。加入4mL叔丁基甲醚经涡旋离心后，提取血浆中的紫杉醇和内标物地西泮，以高效液相色谱仪(LC-2010C，日本岛津)测定各时间点血浆中的紫杉醇含量，测定数据如表8所示。Twelve SD rats (provided by Limin Science and Technology Animal Farm, Qixia District, Nanjing) were randomly divided into 4 groups, with 3 rats in each group, and were given Thai

Kay And paclitaxel albumin nano-preparation (prepared according to the embodiment of the present invention). The dose of 7mg/kg was administered by tail vein injection, and about 0.5mL of blood was collected from the orbit at 5, 15, 30min, 1, 2, 4, 6, 8, 12, 24, and 36h after administration, and placed in the In the centrifuge tube of sodium heparin, centrifuge for 10min (6000r/min) to separate the plasma. Add 4 mL of tert-butyl methyl ether and vortex centrifuge to extract paclitaxel and internal standard diazepam in plasma, and use high performance liquid chromatography (LC-2010C, Shimadzu, Japan) to measure the paclitaxel content in plasma at each time point. The data are shown in Table 8.

Table 8

注射液(P＜0.01)，分别为凯

的1.90和1.79倍。The results showed that this preparation group can significantly prolong the blood circulation time of paclitaxel, which may be due to the soft long-chain structure of polyethylene glycol can significantly reduce the possibility of the carrier being phagocytized by the endothelial reticulum system. In addition, the mean residence time (MRT) and the area under the time-dependent plasma concentration curve (AUC) of this preparation were significantly higher than those of Kai

Injection (P<0.01), Kai

1.90 and 1.79 times.

Embodiment 14 Pharmacodynamic experiments of paclitaxel albumin nanoparticles

样品-3紫杉醇白蛋白纳米制剂(按本发明实施例制备)和样品4-生理盐水，以10mg/kg的剂量由尾静脉注射给药，考察指标如下：40 BALB/c tumor-bearing mice were randomly divided into 4 groups, 10 mice in each group, and the sample 1-Thai Sample 2 - Kay

Sample-3 paclitaxel albumin nano-preparation (prepared according to an embodiment of the present invention) and sample 4-physiological saline are administered by tail vein injection at a dose of 10 mg/kg, and the investigation indicators are as follows:

1. Tumor growth curve: Measure the long diameter a and short diameter b of the tumor with a vernier caliper every two days, calculate the volume of the tumor according to the formula V=0.5×a×b ² , and draw the tumor growth curve;

2. Tumor growth inhibition rate: at the end of the experiment, the mice were sacrificed, the tumor was stripped off, the tumor weight was taken, and the tumor growth inhibition percentage was calculated according to the following formula: inhibition percentage TI (%)=(1-WT/WC)×100%, Where WT is the average tumor weight of the treatment group, and WC is the average tumor weight of the control group;

3. Body weight change curve: the body weight of the mice was measured every day, and the weight change curve of the mice was drawn. Drugs were considered toxic if mice lost more than 20% of their body weight.

和凯

更有效抑制了MDR肿瘤的生长。The results are shown in Table 9 below. The relative tumor growth rate was calculated from the tumor volume at each time point. The results showed that the paclitaxel albumin nano-preparation had

and Kay

More effectively inhibited the growth of MDR tumors.

Table 9

组的肿瘤生长抑制率为60％，凯

组为53％，制剂组为66％。As shown in Table 10, the results of tumor weight after dissection showed that, compared with the average tumor weight of the normal saline group, Thai

The tumor growth inhibition rate of the group was 60%, and Kay

The group was 53%, and the preparation group was 66%.

Table 10

组和本制剂组均未见明显毒性，但在静脉注射泰

后，小鼠出现行动迟缓、呆滞等现象。As shown in Table 11, it can be known from the change of tumor weight (g) that Kai

No obvious toxicity was seen in the group and this preparation group, but in the intravenous injection of Thai

Afterwards, the mice appeared sluggish and sluggish.

Table 11

Example 15 Preparation of Docetaxel Albumin Nanoparticles by High Pressure Homogenization

500 mg of docetaxel was dissolved in 7.0 ml of ethanol. Dissolve 1,300 mg of polyethylene glycol in 1.5 ml of absolute ethanol, heat it in an oil bath at 45°C to completely melt, add the paclitaxel solution into it, stir magnetically until it is completely mixed, remove the ethanol by rotary evaporation, and rapidly dissolve under vigorous stirring. cool down. After drying under vacuum overnight, the solid dispersion was added to 90 ml of human serum albumin solution (4.5% w/v). The mixture was pre-mixed by a high-speed disperser for 1 minute to form a coarse milk, and then transferred to a high-pressure homogenizer. Emulsification was carried out at 9000-40,000 ^psi and the emulsion was recirculated at least 5 times. The obtained system has relatively dense opalescence, and the obtained docetaxel nanoparticles generally have a diameter of 135-200 nm. The result is shown in Figure 7.

The protein nanoparticle suspension was further lyophilized for 48 hours without adding any cryoprotectant. The resulting powder can be easily reconstituted into a protein nanoparticle suspension by adding sterile water or saline. The size of the particles after reconstitution is about the same as before lyophilization.

Example 16 drug loading data

Since the solubility of taxanes in aqueous medium is very low (<1 μg/ml), the amount of free drug in aqueous medium is negligible compared with the amount of drug loaded into the carrier (>2 mg/ml). The amount of the drug loaded into the carrier can be calculated by centrifuging the supernatant and filtering it, and measuring the drug content in the filtrate.

Accurately weigh a certain amount of albumin nano-preparation (prepared according to the embodiment of the present invention) freeze-dried powder, redissolve in double distilled water to the scale, shake and mix. Destroy with methanol and dilute to a certain concentration, measure the content of the drug in the preparation, and calculate the encapsulation efficiency and drug loading according to the following formulas (1) and (2).

The results showed that, as measured by HPLC, the drug loading capacity of paclitaxel albumin nano-preparation freeze-dried powder was (9.67±0.67)%, and the encapsulation efficiency was (94.02±6.4)%. The clinical dosage of paclitaxel is relatively large. The paclitaxel albumin nano-preparation prepared by this method has a high drug loading capacity and encapsulation efficiency, which can reduce the volume of administration and reduce the application of excipient albumin, and has good industrialization prospect.

Example 17

With reference to the method in Example 2, the inventors carried out single-factor comparisons of different water-soluble carriers and proteinaceous substances (except for the differences listed in the table, the rest are the same as in Example 2), and the average particle size of the prepared protein nanoparticles was tested. See Table 13 and Table 14 for details. At the same time, DSC detection showed that the drug existed in the protein nanoparticle preparation in the form of amorphous or solid solution.

Table 13

Table 14

Claims (12)

1. A protein nanoparticle encapsulating taxane drugs, characterized in that the formula of the protein nanoparticle contains the following weight percentages: 0.1-10% taxane drugs, 0.1-40% water-soluble carrier materials, 50-90% protein substances; The proteinaceous material is the following material capable of being cross-linked by sulfhydryl and/or disulfide bonds: naturally occurring or synthetic proteins, synthetic protein polymers, or mixtures thereof; The protein nanoparticles are prepared by the following method: a. Taxanes and water-soluble carrier materials are made into water-soluble carrier solid dispersions containing taxanes. If a solvent-free method is used to prepare the solid dispersion, no organic solvent is introduced. The solvent method does not introduce any other organic solvents except ethanol; b. adding the obtained solid dispersion into an aqueous medium containing protein substances, mixing evenly, and processing the mixture under high shear conditions to obtain a suspension of protein nanoparticles coated with taxane drugs; c. Dry the suspension or first pass through sterile filtration and then dry, and make a solid preparation, semi-solid preparation or gas preparation of protein nanoparticles coated with taxane drugs according to the required dosage form; or The suspension obtained in step b is directly used as a liquid preparation or further prepared into other types of liquid preparations, or the suspension is dried or sterile filtered and then dried before being reconstituted into a liquid preparation. 2. The protein nanoparticle according to claim 1, characterized in that the taxane drugs are selected from one or more of the following substances: paclitaxel and docetaxel. 3. The protein nanoparticle according to claim 1, characterized in that the water-soluble carrier material is selected from one or more of the following substances: polyethylene glycol, polyvinylpyrrolidone, poloxamer, polyethylene Oxides, mixed fatty acid esters, mannitol, urea, sodium alginate, sodium hydroxypropyl cellulose, polyacrylic resin, gelatin, sodium carboxymethyl cellulose. 4. The protein nanoparticle according to claim 1, characterized in that the naturally occurring protein is selected from one or more of the following substances: albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme Enzyme, α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, lipase; the synthetic protein polymer is selected from one of the following substances modified with free sulfhydryl and/or disulfide groups One or more kinds: polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycol, polyglycolide, polycaprolactone or its copolymer, synthetic polyamino acid. 5. the preparation method of protein nanoparticle as claimed in claim 1 is characterized in that the method comprises the following steps: a. Taxanes and water-soluble carrier materials are made into water-soluble carrier solid dispersions containing taxanes. If a solvent-free method is used to prepare the solid dispersion, no organic solvent is introduced. The solvent method does not introduce any other organic solvents except ethanol; b. adding the obtained solid dispersion into an aqueous medium containing protein substances, mixing evenly, and processing the mixture under high shear conditions to obtain a suspension of protein nanoparticles coated with taxane drugs; c. Dry the suspension or first pass through sterile filtration and then dry, and make a solid preparation, semi-solid preparation or gas preparation of protein nanoparticles coated with taxane drugs according to the required dosage form; or The suspension obtained in step b is directly used as a liquid preparation or further prepared into other types of liquid preparations, or the suspension is dried or sterile filtered and then dried before being reconstituted into a liquid preparation. 6. The preparation method according to claim 5, wherein the aqueous medium is selected from water for injection, pure water, mannitol solution, phosphate aqueous solution, dextran solution, glucose aqueous solution, sodium chloride aqueous solution, amino acid solution, vitamin solution, or a mixture of any two or more of them. 7. preparation method according to claim 5 is characterized in that the method for preparing the water-soluble carrier solid dispersion that contains taxanes is melting method, solvent method, solvent-melt method, solvent-spray freeze-drying method, Grinding method, or twin-screw extrusion method; if adopt solvent-free method then do not introduce any organic solvent in these methods, if adopt the method that has solvent then do not introduce any other organic solvents except ethanol; described high shear The condition refers to the application of equipment with high pressure and high shear force at ^the same time, so that the mixture can be efficiently mixed, pulverized, dispersed and emulsified; wherein, high pressure refers to a pressure range of 5,000 to 30,000 psi. 8. The preparation method according to claim 7, characterized in that the method for preparing the water-soluble carrier solid dispersion containing taxanes is a solvent-melting method. 9. The preparation method according to claim 7, characterized in that high pressure refers to a pressure range of 6000-25000 ^psi . 10. The preparation method according to claim 5, characterized in that the protein nanoparticles have an average particle diameter of 20 to 1000 nm 11. The preparation method according to claim 10, characterized in that the average particle diameter of the protein nanoparticles is 20-200 nm. 12. The preparation method according to claim 5, characterized in that the drying method adopts freeze drying or spray drying; the sterile filtration adopts 0.22 μm filter.

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