Fast-dissolving nanoparticle composition and method for producing same

Abstract

The present invention provides a fast-dissolving nanoparticle composition and its preparation method. The composition comprises an active ingredient, albumin and a particle stabilizer, and optionally comprises a lyoprotectant. The active ingredient has the characteristics of being insoluble or poorly soluble in water, and soluble or easily soluble in a specific organic solvent, preferably paclitaxel or docetaxel. The composition of the present invention dissolves quickly, does not require complicated operations, has less foaming, has better stability, and causes less allergic reactions.

Description

[Technical field]

[0001] The present invention relates to the pharmaceutical field, and more particularly to a rapidly dissolving nanoparticle composition comprising human serum albumin and a hydrophobic drug, and a method for preparing the same. [Background technology]

[0002] Paclitaxel is an anticancer drug extracted from the trunk and bark of Taxol plants. It is a mitotic microtubule inhibitor that has the effect of polymerizing and stabilizing intracellular microtubules. In the mitotic phase, paclitaxel impairs the scission of microtubules and blocks cells in the G2 and M phases of the cell cycle. Thus, paclitaxel immobilizes rapidly dividing tumor cells in the mitotic phase, blocking cell replication and causing death. Paclitaxel has significant clinical activity for various cancers (e.g., breast cancer, ovarian cancer, lung cancer, bladder cancer, etc.).

[0003] However, its poor water solubility makes it difficult to apply to the human body. To make paclitaxel suitable for intravenous injection, Bristol-Myers Squibb (BMS) developed Taxol, which uses a surfactant, polyoxyethylene castor oil (Cremophor EL), together with absolute ethanol as a solvent to increase the solubility of paclitaxel. Taxol has significant activity against ovarian, breast, lung, esophageal, and head and neck cancers. However, it has been shown that Taxol can induce administration-related toxicity as well as significant acute and cumulative toxicities, such as bone marrow suppression, neutropenic fever, and allergic reactions. These side effects are associated with the surfactant polyoxyethylene castor oil used (Anantbhushan et al., Asia Pac J Clin Oncol. 2013;9:176-181). Clinical study reports and post-marketing safety data indicate that the overall incidence of allergic reactions in Taxol was about 39%. Currently, when using Taxol, patients must also take antihistamines and steroid hormones to counteract side effects caused by the surfactant.

[0004] US5439686, US6537579, US6749868, US2006121119, CN97199720.9, CN03108361.7 and CN200610077006.4 etc. disclose a method for preparing paclitaxel albumin nanoparticles, which provides a formulation with significant advantages and develops the marketed drug Abraxane®, which shows the following advantages: 1) without pretreatment with antihistamines before administration, the allergy incidence rate is significantly lower than Taxol, and when combined with PD-1 / PDL1, the activity of PD-1 / PDL1 is not affected; 2) the injection time is shortened from 3 hours of the normal preparation to 30 minutes; 3) after blood injection, it is quickly disintegrated and dispersed, and transported to tumor tissues by a specific transport mechanism, improving safety and efficacy.

[0005] However, this system still has many shortcomings, for example, the formulation procedure before using lyophilized powder injection albumin-bound paclitaxel Abraxane® includes the following six steps: 1) dissolve and disperse with 20ml of 0.9% sodium chloride injection per vial under aseptic technique; 2) use a sterile syringe to slowly inject 20ml of 0.9% sodium chloride injection along the inner wall of the vial for at least 1 minute; 3) do not inject 0.9% sodium chloride injection directly into the lyophilized material / powder to avoid foaming; 4) after the injection is completed, leave the vial to stand for at least 5 minutes to allow the lyophilized mass / powder to completely penetrate; 5) gently shake the vial or slowly turn the vial upside down for at least 2 minutes to completely disperse and dissolve all the lyophilized mass / powder in the vial and avoid foaming; and 6) leave the vial to stand for 15 minutes until the foaming disappears if foaming occurs. According to this standard operation, it takes at least 8 minutes, and if foam is accidentally generated, it takes more than 23 minutes. This prescribing operation before clinical use is very complicated, and the prescriber's operation is likely to be inappropriate, the dispersion time of the product is long, the risk of contamination during pharmaceutical preparation is high, and the space and manpower required for the liquid distribution operation are required, which affects the work efficiency of the prescriber. In addition, if the liquid preparation operation is not appropriate, there is a lot of foaming and the dissolution is likely to be incomplete, which leads to inaccurate measurement of the drug solution and clogging of the liquid delivery pipe, which may cause problems such as impaired administration safety and medical trouble.

[0006] In addition, this system contains a large amount of human serum albumin (HSA), which has a high potential to cause allergies. The source of HSA is human blood, and there is a possibility of contamination during blood collection, storage, etc., raising concerns about the safety of blood products. In addition, HSA is expensive and in short supply in some areas.

[0007] Patent application 201580045838.5 filed by the applicant in 2015 provides purified therapeutic nanoparticles consisting of an active ingredient and human serum albumin, the weight ratio of the human serum albumin to the active ingredient being selected from the range of 0.01:1-8.5:1, and the therapeutic nanoparticles being substantially free of free HSA not contained in the nanoparticles. In some embodiments, when the pharmaceutical composition is provided as a lyophilized powder, it contains one or more lyophilization excipients selected from mannitol, sucrose, lactose, maltose, trehalose, dextran, or combinations thereof. The therapeutic nanoparticles disclosed in this patent are desired to be administered by intravenous injection, and therefore sterilization of the product must be ensured. Both the nanoparticles and human serum albumin are temperature sensitive, and therefore cannot be sterilized by heat sterilization, and possible sterilization means include aseptic manufacturing or sterile filtration of the formulation. In this case, the suspension obtained after removing the organic solvent is sterilized by filtration through a membrane filter, and then freeze-dried to obtain a solid. The solid was suspended in water. In some embodiments, the solid is suspended in water such that the paclitaxel is adjusted to about 5 mg / ml.

[0008] In 2011, Ismael B et al. reported a method to increase the solubility of paclitaxel by pretreating the HSA solution to pH 2.7 to expose the hydrophobic ends of the protein and strengthen the binding with paclitaxel, then slowly adding paclitaxel dissolved in an organic solvent and adjusting the pH to 7.0. They reported that NaCl (0, 0.2M, 0.4M, 0.6M, 0.8M and 1.0M) was added during the manufacturing process, and it was found that when NaCl was 0-0.8M (equivalent to 0-4.6wt%), it had a solubilizing effect on paclitaxel, and when NaCl was 0.4M (equivalent to 2.3wt%), the binding rate of paclitaxel to HAS was the highest at 91.0%. In this method, the added NaCl was not removed from the system. This report does not concern research into the urgent issues of the prior art, such as whether nanoparticles are contained in the system, freeze-drying the system, and consideration of stability before and after freeze-drying and reconstitution time. (Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes[J]. International Journal of Nanomedicine, 2011, 2011:1193-1200.) Summary of the Invention

[0009] The composition of the present invention can shorten the redissolution time of the formulation to within 2 minutes, reduce the foaming phenomenon, and avoid problems such as incomplete dissolution. Furthermore, the functional structure and stability of the nanoparticles are not changed, and the safety and therapeutic effect are maintained. Furthermore, the amount of HSA used in the fast-dissolving nanoparticle composition can be reduced, which can reduce allergic reactions caused by HSA.

[0010] Compared with the 2011 study by Ismael B et al., the preparation method of the present invention involves the formation of nanoparticles without the need to adjust the pH of the system. In addition, the inventors unexpectedly found that not only is an appropriate concentration of NaCl necessary for the preparation of fast-dissolving nanoparticle compositions, but also the content of NaCl in the final fast-dissolving nanoparticle compositions needs to be controlled.

[0011] Explanation of terms As used herein, the term "nanoparticle" refers to a particle that is nanometer in at least one dimension (eg, one, two, or three dimensions), for example, on the order of about 1 nm, about 10 nm, or about 100 nm.

[0012] As used herein, the term "purified nanoparticles" refers to nanoparticles formed from human serum albumin and an active ingredient, in which free human serum albumin has been removed by 5%-50% of the original amount.

[0013] As used herein, the term "rapidly dissolving nanoparticle composition" refers to a nanoparticle composition obtained by freeze-drying the purified nanoparticles described above, which disperses rapidly upon addition of a solvent.

[0014] As used herein, the term "about" refers to 10% above or below a particular value. For example, "about 50 nm" refers to 45 nm-55 nm.

[0015] The term "human serum albumin monomer" or "HSA monomer" refers to a water-soluble globulin consisting of 585 amino acids with an approximate molecular weight of about 66,000 daltons. It is the most abundant protein in human plasma. In size exclusion chromatography, human serum albumin monomer has the longest retention time and accounts for the majority of albumin products in normal humans. HSA has multiple hydrophobic binding sites that can bind a diverse set of drugs, especially neutrally and negatively charged hydrophobic compounds.

[0016] The term "active ingredient" as used herein means a pharma- ceutical active ingredient. Specifically, an active ingredient means any substance or entity capable of playing a therapeutic role (e.g., treating, preventing, ameliorating, or suppressing any disease and / or disorder).

[0017] The term "dialysis factor" refers to the ratio of the volume of dialysate consumed to the volume of sample solution in a dialysis treatment, where the volume of sample solution is kept constant.

[0018] The term "lyoprotectant" refers to a substance added to a pharmaceutical composition containing purified nanoparticles to protect the nanoparticles during the freeze-drying process.

[0019] The terms "treat" and "treatment" refer to the medical management of a patient's disease, disorder, or condition (see, e.g., Stedman's Medical Dictionary). "Treating cancer" means reducing the number of symptoms of cancer, lessening the severity of one or more symptoms, or slowing the progression of cancer.

[0020] A "therapeutically effective amount" of a particular therapeutic agent means that amount of the agent sufficient to reduce the number of symptoms of cancer, lessen the severity of one or more symptoms, or slow the progression of cancer.

[0021] In a first aspect, the present invention provides a fast-dissolving nanoparticulate composition comprising an active ingredient, albumin, and a particle stabilizer, and optionally a lyoprotectant.

[0022] In some embodiments, the present invention provides a fast-dissolving nanoparticle composition comprising an active ingredient, an albumin, a lyoprotectant, and a particle stabilizer.

[0023] The active ingredient is selected from active ingredients suitable for being encapsulated in human serum albumin and should have the properties of being insoluble or poorly soluble in water and soluble or readily soluble in a particular organic solvent.

[0024] In some embodiments, the active ingredient is selected from taxanes (including but not limited to paclitaxel, docetaxel, cabazitaxel, hydrophobic derivatives of docetaxel), macrolides (including but not limited to rapamycin and its derivatives, epothilone B and its derivatives, tanespimycin and its derivatives, etc.), camptothecins (including but not limited to 10-hydroxycamptothecin, SN-38 and its derivatives, etc.), anthracyclines (including but not limited to aclacinomycin and pirarubicin), or other active ingredients (including colchicine and its derivatives, thiocolchicine dimer, amiodarone, liothyronine, cyclosporine, exemestane, flutamide, fulvestrant, romidepsin, semustine, ibuprofen, etc.).

[0025] In some embodiments, the active ingredient is selected from a taxane, preferably the taxane is selected from paclitaxel or docetaxel.

[0026] The albumin is selected from serum albumins having carrier action, such as human serum albumin and bovine serum albumin, preferably human serum albumin.

[0027] The lyoprotectant is selected from mannitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, preferably a combination of mannitol and sucrose, the ratio of the two being preferably 10:1-1:1, preferably 8:1-2:1, preferably 8:1-3:1, more preferably 5:1-2:1, more preferably 5:1-3:1, most preferably 5:1.

[0028] The particle stabilizer is selected from sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, preferably sodium chloride.

[0029] In some embodiments, the fast dissolving nanoparticle composition comprises, by weight of each component, 1-30% active ingredient, 0.1-30% protein, 0.00001-3% particle stabilizer, and the remainder lyoprotectant.

[0030] In some embodiments, the fast-dissolving nanoparticle composition has a weight percent of the particle stabilizer of 0.00001-3%, preferably 0.0001-1%, or any subrange therein, such as 0.0001-1%, 0.0001-0.1%, 0.0005-0.5%, 0.001-0.1%, 0.005-0.05%, 0.008-0.022%, etc.

[0031] In some embodiments, the fast-dissolving nanoparticle composition has a weight percent of the active ingredient of 1-30%, preferably 2-20%, or 3-15%, or 5-10%.

[0032] In some embodiments, the fast-dissolving nanoparticle composition has a protein content by weight of 0.1-30%, or 1-25%, or 5-20%.

[0033] In some embodiments, the binding rate between the protein and the active ingredient is greater than 90%.

[0034] In said composition the weight ratio of protein to active ingredient is 0.1:1-5:1, preferably 0.3:1-3:1 or 0.5:1-3:1 or 1:1-3:1 or 0.5:1-2.5:1. For example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.70:1, 0.8:1, 0.9:1, 0.95:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, or a range selected between any two of the above ratios. In some specific embodiments, the protein is human serum albumin.

[0035] In some embodiments, the composition has an average particle size of 30-200 nm, preferably 100-180 nm, preferably 110-170 nm, more preferably 110-160 nm, more preferably 110-140 nm, even more preferably 115-130 nm, even more preferably 118-125 nm, and most preferably 118-122 nm, e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 111, 112, 113, 114, 115, 1116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 , 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 170, 175, 180, 185, 190, 195, 200 nm, or a range selected between any two of the above values.

[0036] In some embodiments, the active ingredient is dissolved using an organic solvent. Those skilled in the art can select an appropriate organic solvent according to the nature of the active ingredient. The organic solvent is selected from a pure solvent with low water solubility and low boiling point, or a mixed solvent with small molecule alcohols. Preferably, the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, or tert-butanol, preferably chloroform or a combination of chloroform and ethanol. The present invention uses any suitable method to remove the organic solvent in the emulsion, for example, by means of vacuum evaporation, dialysis, etc., to minimize the residual organic solvent in the product. In the fast-dissolving nanoparticle composition, the residual amount of the organic solvent is less than 0.1 mg / ml, preferably less than 0.08 mg / ml, 0.05 mg / ml, 0.04 mg / ml, 0.03 mg / ml, 0.02 mg / ml or 0.01 mg / ml, more preferably less than 5 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml or 0.01 μg / ml.

[0037] In some embodiments, the fast-dissolving nanoparticle composition does not contain any surfactants, such as polyoxyethylene castor oil, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylmethylcellulose, poloxamer, lecithin, or cholate.

[0038] In a second aspect, the present invention provides a pharmaceutical composition comprising a rapidly dissolving nanoparticle composition according to the first aspect.

[0039] In some embodiments, the pharmaceutical composition is in solid or liquid form.

[0040] In some embodiments, the pharmaceutical composition is provided in a liquid form, including, but not limited to, a form suitable for administration by injection to a subject, hi certain embodiments, the pharmaceutical composition is an injectable.

[0041] In some specific embodiments, the pharmaceutical composition is provided in a liquid form with the fast-dissolving nanoparticles suspended in a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, buffers, preservatives, water for injection, saline, and isotonic solutions. In certain embodiments, the liquid form pharmaceutical composition of the present invention contains an active agent (e.g., paclitaxel) in an amount of 0.1-100mg / ml, preferably 0.5-50mg / ml, more preferably 1-20mg / ml, e.g., 5mg / ml.

[0042] In another embodiment, the pharmaceutical composition is in a solid form, including but not limited to, a dry powder or a lyophilized powder.

[0043] In some specific embodiments, the present invention provides a pharmaceutical composition in solid form. The fast-dissolving nanoparticle composition has an active drug (e.g., paclitaxel) content of 1-30% by weight, preferably 2-20% or 3-15% or 5-10%. The pharmaceutical composition in solid form can be resuspended in a pharma- ceutical acceptable carrier, including but not limited to buffers, preservatives, water for injection, saline, and isotonic solutions. In certain embodiments, the solid is suspended in water for injection, and preferably the resulting resuspension has an active drug (e.g., paclitaxel) content of about 5 mg / ml.

[0044] In a third aspect, the present invention provides a method for producing the fast-dissolving nanoparticle composition. The method of the present invention includes the steps of (1) dissolving an active ingredient in an organic solvent to prepare an oil phase, and dissolving a protein in water to prepare an aqueous phase, (2) mixing and homogenizing the oil phase and the aqueous phase to form a nanoemulsion, (3) removing the organic solvent in the nanoemulsion to obtain a suspension, and (4) dialysis, and optionally (5) freeze-drying.

[0045] In some embodiments, in step (1), the organic solvent is selected from a low water solubility, low boiling point pure solvent, or a mixed solvent with a small molecule alcohol. Preferably, the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, or tert-butanol, preferably chloroform or a combination of chloroform and ethanol. Those skilled in the art can select a suitable organic solvent according to the nature of the active ingredient. In certain embodiments, when the active ingredient is a taxane, a useful organic solvent is selected from one or more of chloroform and ethanol. More specifically, when the active ingredient is paclitaxel or docetaxel, the organic solvent can be a mixed system of chloroform and ethanol. In certain embodiments, the volume ratio of chloroform to ethanol is 1:1-20:1, preferably 1:1-15:1, preferably 1:1-11:1, for example, selected from 1:1, 4:1, 9:1, and 11:1.

[0046] In certain embodiments, the concentration of the active ingredient in the oil phase is in the range of 20-500mg / ml, preferably 50-350mg / ml, preferably 60-250mg / ml, preferably 80-200mg / ml. In certain embodiments, the concentration of the active ingredient in the oil phase is selected from 20mg / ml, 30mg / ml, 50mg / ml, 83mg / ml, 100mg / ml, 133mg / ml, 150mg / ml, 167mg / ml, 180mg / ml, 200mg / ml, 250mg / ml, 300mg / ml, 333mg / ml, 450mg / ml and 500mg / ml.

[0047] In certain embodiments, the concentration of the protein in the aqueous phase is between 2% and 10% (w / v), preferably between 2% and 7% (w / v), preferably between 2% and 5% (w / v), e.g. selected from 2%, 4%, 5% and 10%.

[0048] In some embodiments, step (2) comprises mixing an oil phase and an aqueous phase to form a nanoemulsion, the volume ratio of the oil phase to the aqueous phase being 1:10-1:100, preferably 1:10-1:60, more preferably 1:20-1:40. In some embodiments, the mixing ratio of the oil phase to the aqueous phase is 3:100 or 1:25. The nanoemulsion is formed using methods known in the art, including but not limited to homogenization methods. For example, the mixture of the oil phase and the aqueous phase is homogenized in a high shear disperser, and then homogenized in a high pressure homogenizer to obtain an oil-in-water emulsion. In a specific embodiment, the mixture of the oil phase and the aqueous phase is homogenized in a high shear disperser for 2-10 minutes, and then homogenized in a high pressure homogenizer at 10,000-20,000 psi to obtain an oil-in-water emulsion.

[0049] In some embodiments, the organic solvent in the nanoemulsion in step (3) can be removed by any suitable method known in the art, such as vacuum evaporation, dialysis, etc. Here, vacuum evaporation includes a falling film evaporator.

[0050] In some embodiments, the organic solvent in the nanoemulsion is removed using falling film evaporation. In a specific embodiment, the emulsion is subjected to a falling film evaporator at 60° C. and a vacuum of 40 mbar to remove the organic solvent in the emulsion. After removing the organic solvent, the resulting suspension contains the nanoparticles of the present disclosure. However, the suspension in this case also contains excess albumin, which is not actually involved in the formation of the nanoparticles.

[0051] In certain embodiments, the organic solvent in the nanoemulsion is removed using dialysis, in which case step (3) may be performed in conjunction with step (4).

[0052] In some embodiments, in step (4), dialysis is performed at a factor of 2-10, preferably 3-6.

[0053] In a particular embodiment, the dialysis method comprises adding a particle stabilizer to the suspension obtained in step (3) to a concentration of the particle stabilizer in the suspension of 0.01%-1.4% (w / v), and dialyzing with an aqueous solution of a lyoprotectant. The concentration of the particle stabilizer in the suspension is preferably 0.1-1.4% (w / v), and more preferably 0.1-0.9% (w / v). The aqueous solution of a lyoprotectant preferably contains 1%-10%, preferably 2%-8%, and more preferably 5%-6% of the lyoprotectant.

[0054] In a particular embodiment, the dialysis method is to perform dialysis with an aqueous solution of a lyoprotectant, said aqueous solution further comprising a particle stabilizer, said particle stabilizer having a concentration of 0.01%-1.4% (w / v), preferably 0.05%-0.9% (w / v), preferably 0.05%-0.5% (w / v), more preferably 0.05%-0.15%. Preferably, the aqueous solution of a lyoprotectant comprises 1%-10%, preferably 2%-8%, preferably 5%-6% of the lyoprotectant.

[0055] In some embodiments, the dialysis method is to perform dialysis with a particle stabilizer solution of suitable concentration, the particle stabilizer solution having a concentration of 0.01%-1.4% (w / v), preferably 0.1%-1.4% (w / v), preferably 0.1%-0.9% (w / v).After dialysis, it is optional to add a lyoprotectant, preferably so that the resulting liquid contains 1%-10%, preferably 2%-8%, more preferably 5%-6% of the lyoprotectant.

[0056] In some embodiments, the conditions for lyophilization in step (4) are freezing at between -20°C and 60°C and drying at a temperature between 0°C and +40°C.

[0057] Since the fast-dissolving nanoparticles of the present invention are preferably administered by intravenous infusion, it is necessary to ensure the sterility of the product. Both the nanoparticles and serum albumin are temperature sensitive and therefore cannot be sterilized by heat sterilization, and possible sterilization protocols include aseptic manufacture or sterile filtration of the formulation.

[0058] In a fourth aspect, the present invention also provides a method for using the above-mentioned fast-dissolving nanoparticles or pharmaceutical compositions. Such fast-dissolving nanoparticles or pharmaceutical compositions can effectively transport various active ingredients, and are therefore useful for treating diseases that respond to the active ingredients. For example, the fast-dissolving nanoparticles or compositions thereof are useful for treating liver cancer, prostate cancer, lung cancer, breast cancer, multiple myeloma, transplant rejection, colon cancer, and lymphoma cancer. Other diseases include fever, etc.

[0059] In a fifth aspect, the present disclosure also provides the use of the above-mentioned fast-dissolving nanoparticles or pharmaceutical composition in the manufacture of a medicament for the treatment of a disease that responds to the active ingredient.For example, the disease includes liver cancer, prostate cancer, lung cancer, breast cancer, multiple myeloma, transplant rejection, colon cancer, lymphoma cancer.Other diseases include fever, etc.

[0060] Unless otherwise defined, all scientific terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0061] Although the numerical ranges and approximate parameter values ​​are described in broad ranges in the present invention, all numerical values ​​in the specific examples are described as precisely as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation in each measurement. Also, all ranges disclosed herein should be understood to include all and any subranges contained therein. For example, as described herein, the range "1-10" includes all and any subranges between the minimum value of 1 and the maximum value of 10 (including the end points), i.e., all subranges beginning with a minimum value of 1 or greater, such as 1-6.1, and all subranges ending with a maximum value of 10 or less, such as 5.5-10. Additionally, all references stated to be "incorporated herein" are understood to be incorporated in their entirety.

[0062] It should be noted that the singular forms include plural referents when used in this disclosure. Unless the context clearly dictates otherwise, the terms "or" and "and / or" are used interchangeably.

[0063] It will be understood by those skilled in the art that particle size can be determined using any suitable method now existing or that will become apparent in the future, including but not limited to gravitational settling, sieving, microscopic observation, or a laser particle sizer. It will also be understood that when the therapeutic nanoparticles disclosed in this disclosure comprise multiple particles, the particles do not necessarily all have to have the same particle size, but are also within the scope of this disclosure as long as their average particle size is within the above range. In some specific embodiments, particle size is determined by a laser particle sizer.

[0064] The rapidly dissolving nanoparticles or pharmaceutical compositions of the present invention have one or more of the following advantages:

[0065] (1) It has a fast dissolution rate and produces little foaming. The preparation of albumin-bound paclitaxel Abraxane®, which is already on the market, requires complicated operations, takes at least 8 minutes, has a long product dispersion time, and includes six steps that increase the risk of contamination during pharmaceutical preparation; requires a long space and manpower for the liquid distribution operation, which affects the work efficiency of the compounder; if the liquid preparation operation is not performed properly, there will be a lot of foaming and incomplete dissolution, which will lead to inaccurate measurement of the drug solution and clogging of the liquid delivery pipes, which may compromise the safety of medication or lead to medical troubles.

[0066] The freeze-dried product of the present invention can be dissolved quickly and does not require complicated operations. The redissolution time of the formulation can be shortened to within 2 minutes, even to within 1 minute, and the foaming phenomenon can be reduced, and problems such as incomplete dissolution can be avoided. This greatly improves the convenience and working efficiency of clinical application and is advantageous in ensuring the safety of drugs.

[0067] (2) Good stability. The rapidly dissolving nanoparticles or pharmaceutical composition of the present invention have good stability during the preparation process and after redissolution and resuspension, and even when left at room temperature for 24 or 48 hours, there is no significant change in liquid properties, particle size, light transmittance, etc. The rapidly dissolving nanoparticles or pharmaceutical composition were left under accelerated conditions for 10 days, but there was no significant change in particle size, light transmittance, redissolution time, etc.

[0068] (3) Improving safety The present invention optimizes the formulation compared to the conventional technology, significantly reducing the amount of human serum albumin used and significantly reducing the allergic reaction caused by HSA while maintaining the functional structure and stability of the nanoparticles. The product of the present invention has no significant difference in therapeutic effect compared to Abraxane (registered trademark), and its safety has been significantly improved.

[0069] (4) The production method of the present invention is simple and easy to carry out, and is suitable for mass production. [Brief description of the drawings]

[0070] [Figure 1] FIG. 1 shows the mean concentration-time curves (Mean±SD, N=24) of total paclitaxel in plasma of Beagle dogs following administration of lyophilized powder 4 and Abraxane. [Diagram 2] FIG. 2 shows the mean concentration-time curves (Mean±SD, N=24) of free paclitaxel in plasma of Beagle dogs following administration of lyophilized powder 4 and Abraxane. [Diagram 3] FIG. 3 shows the results of tissue distribution after tail vein injection of 20 mg / kg of lyophilized powder 4 and Abraxane® in mice bearing human breast cancer JIMT-1. [Figure 4] FIG. 4 shows the inhibitory effect of drugs on human breast cancer JIMT-1 xenograft tumors (* p < 0.05, *** p < 0.001, compared to vehicle control). [Diagram 5] FIG. 5 shows the drug inhibitory effect on human breast cancer KPL-4 xenograft tumors (*** p < 0.001 compared to vehicle control). [Figure 6] FIG. 6 shows the inhibitory effect of the drug on human pancreatic cancer CFPAC-1 xenograft tumors (*** p < 0.001 compared to vehicle control). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] Detection method (1) Measurement method of the content of each ingredient in the product 1. Measurement of human serum albumin content The HPLC method was used. The albumin content was calculated by the external standard method using a UV detector equipped with a Tosohaas TSK G3000 SWXL gel column, with a detection wavelength of 228 nm, a mobile phase of 0.1 mol / L dipotassium hydrogen phosphate solution (adjusted to pH 7.0 ± 0.1 with hydrochloric acid), a column temperature of 25°C, a flow rate of 0.7 ml / min, and a sample injection volume of 10 μL. Preparation of sample solution: The test solution was diluted with 0.9% sodium chloride solution so that the albumin concentration was less than 3 mg / ml to prepare a sample solution.

[0072] 2. Measurement of Paclitaxel Content The HPLC method was used. The content of paclitaxel was calculated by the external standard method using an ultraviolet detector equipped with an octadecylsilane-bonded silica gel column, with a wavelength of 228 nm, a mobile phase of acetonitrile-water (1:1, v / v), a column temperature of 25°C, a flow rate of 1.0 ml / min, and a sample injection volume of 10 μl. Preparation of sample solution: Paclitaxel was diluted with acetonitrile until it was completely dissolved, to prepare a sample solution with a concentration of 20 to 200 μg / ml.

[0073] 3. Measurement of mannitol content The HPLC method was used. The mannitol content was determined using a differential refractive index detector equipped with a Sepax Carbomix Ca-NP column. The mobile phase was water, the flow rate was 0.5 ml / min, the column temperature was 50°C, the detection temperature was 55°C, the differential refractive index detector was used, and the sample injection volume was 20 μl. The amount of mannitol was calculated using the external standard method.

[0074] 4. Measurement of sucrose content An HPLC method was used. The sucrose content was calculated using an evaporative light scattering detector with an Agilent ZORBAX NH2 column. The mobile phase was acetonitrile-water (80:20, v / v), with a flow rate of 1.5 ml / min, column temperature of 35°C, and an evaporative light scattering detector with a nebulizer temperature of 55°C, drift tube temperature of 55°C, gas flow rate of 1.0 ml / min, and sample injection volume of 5 μL, using a standard curve.

[0075] 5. Measurement of sodium ion content Ion chromatography was performed using a conductance detector equipped with a cation exchange column (Dionex IonPacTM CS16). The mobile phase was 30 mM methanesulfonic acid aqueous solution, the flow rate was 0.4 ml / min, the column temperature was 30°C, and the sample injection volume was 25 μL. The sodium ion content was calculated by the external standard method.

[0076] Detection method (2) Transmittance measurement method The change in transmittance of a light-transmitting product (transmitted light intensity ≧0.2%) can reflect the change in stability of the product, and the smaller the change in transmittance within a certain period of time, the better the stability. It can be seen that a decrease in transmittance reflects particle aggregation, and a high transmittance reflects the settling of particles.

[0077] This measurement was performed using a multiple scattering light analyzer (Turbiscan, Formulaaction). The product was scanned at different times and the change in the product transmittance or backscattered light was monitored to determine whether the product was experiencing instability phenomena such as aggregation, settling, or delamination.

[0078] Preparation of sample cell: The sample to be measured was shaken and placed in the sample cell. The sample volume was about 20 ml. The sample was set on the sample stand and covered with the sample cell. Sample measurement: Using Smart Scan, the scan time was set to 24 hours, and automatic measurement was performed every 30 seconds to obtain the transmittance values ​​at a fixed position and at different times.

[0079] The following examples are intended to more clearly disclose the compositions of the present invention and are not intended to limit any aspect of the present disclosure. The reagents and devices used in the examples were of the same model number and / or manufacturer.

[0080] Example 1: Screening of particle stabilizer concentrations during preparation 20 g of paclitaxel (CAS: 33069-62-4, Hainan Yew Pharmaceutical Co., Ltd.) was dissolved in 120 ml of chloroform / ethanol (11:1, v / v) to prepare the oil phase solution, and 800 ml of human serum albumin solution (CAS: 70024-90-7, Hebei Daan Pharmaceutical Co., Ltd.) (20% w / v) was weighed out and filled with water up to 4 L to prepare the aqueous phase solution, and the oil phase and aqueous phase were emulsified online using a high shear disperser (model number F22E, FLUKO Equipment (Shanghai) Co., Ltd.) to form the primary emulsion. A high-pressure homogenizer (model number M-110EH30K, Microfluidics Co., Ltd.) was used to homogenize at 10,000-20,000 psi to obtain a nanoemulsion, which was then transferred to a falling film evaporator (model number APLS-70L, Shanghai Tofflon Science and Technology Co., Ltd.) with a heating temperature of 50°C and reduced pressure evaporation at a vacuum of less than 80 mbar to remove the organic solvent in the solution. The resulting paclitaxel albumin nanoparticles had an average diameter of 114 nm and were a translucent suspension.

[0081] The resulting suspension was dialyzed using a 300 kD ultrafiltration membrane, and was dialyzed six times with each of the dialysis substances in the table below, resulting in the replacement of most of the free human serum albumin in the suspension. Purified nanoparticle suspensions 1-1 to 1-8 were obtained.

[0082] The purified nanoparticle suspension was left at room temperature for 24 hours while measuring the transmittance using the above-mentioned "Detection Method (ii) Transmittance Measurement Method", and the appearance was observed, and the results were as follows.

[0083] [Table 1]

[0084] As a result, no precipitation occurred after dialysis using 0.1-1.4% sodium chloride solution or after leaving it for 24 hours, and it was confirmed that there was no significant change in the transmittance of the product compared to the taxanol albumin nanoparticles before dialysis, indicating that a certain range of sodium chloride concentration was useful for stabilizing the product. In particular, 0.9% sodium chloride solution was the best, and too high a NaCl concentration was disadvantageous for stabilizing the particles.

[0085] Example 2 Preparation of sodium chloride-containing fast-dissolving nanoparticles and stability test (different dialysis ratios) 20 g of paclitaxel was dissolved in 200 ml of chloroform / ethanol (11:1, v / v) to prepare an oil phase solution, 800 g of human serum albumin solution (20% w / v) was weighed out, and water was added up to 4 L to prepare an aqueous phase solution. The oil phase and aqueous phase were emulsified online using a high shear disperser to form a primary emulsion. Homogenization was performed using a high pressure homogenizer at 10,000 - 20,000 psi to obtain a nanoemulsion, which was transferred to a falling film evaporator, set at a heating temperature of 50 °C, and reduced pressure evaporation was performed at a vacuum of less than 80 mbar to remove the organic solvent in the solution. The resulting paclitaxel-albumin nanoparticles had an average diameter of 112 nm and were a translucent suspension.

[0086] The obtained suspension was dialyzed using a 300kD ultrafiltration membrane, and 5% mannitol solution (containing 0, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% sodium chloride, respectively) was used as the dialysis solution, and dialyzed at four times the volume. After the dialysis was completed, most of the free human serum albumin in the suspension was replaced. Purified nanoparticle suspensions 2-0 - 2-6 were obtained. 20 ml of each purified nanoparticle suspension was taken, placed in an ampoule, and freeze-dried, obtaining freeze-dried powders of fast-dissolving nanoparticles 2-0 - 2-6 as white cakes. The accelerated stability of the freeze-dried powder was examined and the results are shown in Table 2.

[0087] The freeze-dried nanoparticle powders 2-0 - 2-6 obtained above were each added to 20 ml of water for injection, and the vial was gently shaken to completely disperse and dissolve all the freeze-dried cakes / powder in the vial. All the white cakes were completely dissolved in about 1 minute. The particle size of the suspension after resuspending the freeze-dried powders 2-1 - 2-6 did not change significantly compared to the nanoparticle suspension before freeze-drying (particle size of 112 nm). The redissolved particle size of sample 2-0 after freeze-drying became large at 137 nm. The redissolved suspension of the above samples was left at room temperature for 24 hours, but no significant changes were observed in the solution appearance and transmittance. It was suggested that the addition of sodium chloride during dialysis is advantageous for the stability of particle size during redissolution of freeze-drying.

[0088] The freeze-dried powders 2-0 - 2-6 were left at 60°C for 5 days, and the white cake was completely dissolved within 2 minutes. The redissolved samples were left at room temperature for 24 hours, and there was no significant change in the appearance of any of the samples. After 48 hours of standing, no precipitation was observed in samples 2-1 and 2-2.

[0089] [Table 2]

[0090] Example 3 Screening of different lyoprotectants 20g of paclitaxel was dissolved in 200ml of chloroform / ethanol (11:1, v / v) to prepare an oil phase solution, 800ml of human serum albumin solution (20% w / v) was weighed out, and water was added up to 4L to prepare an aqueous phase solution. The oil phase and aqueous phase were emulsified online using a high shear disperser to form a primary emulsion. The nanoemulsion was homogenized at 10000-20000psi using a high pressure homogenizer to obtain a nanoemulsion, which was transferred to a falling film evaporator, set at a heating temperature of 50°C, and evaporated under reduced pressure at a vacuum of less than 80mbar to remove the organic solvent in the solution. The resulting paclitaxel-albumin nanoparticles had an average diameter of 122nm and were a translucent suspension.

[0091] The obtained suspension was adjusted to a concentration of 0.9% by adding an appropriate amount of sodium chloride. The suspension was dialyzed using a 300 kD ultrafiltration membrane, and purified nanoparticle suspensions 3-1 to 3-5 were obtained by dialysis using five types of dialysis fluid containing different lyoprotectants as shown in the table below at three times the volume. No significant change in particle size was observed during dialysis.

[0092] The purified nanoparticle suspensions 3-1 - 3-5 were each separated and freeze-dried in a freeze dryer for 40 hours to obtain stable white cakes, which were designated as the freeze-dried powders of fast-dissolving nanoparticles 3-1 - 3-5.

[0093] The freeze-dried powders of the fast-dissolving nanoparticles 3-1 - 3-5 were resuspended in water to prepare isotonic solutions, and the white cake was completely dissolved within 2 minutes and did not change significantly even after 24 hours at room temperature. For the freeze-dried samples dialyzed against a 5% mannitol + 1% sucrose solution, the suspension after resuspension was more stable at room temperature and did not change significantly even after 48 hours.

[0094] [Table 3]

[0095] The above freeze-dried nanoparticle powders 3-1 to 3-5 were left at a high temperature of 60°C for 10 days, and the transmittance was measured according to "Measurement method (2) Transmittance measurement method", and the average particle size was measured. The results are shown in the table below.

[0096] [Table 4]

[0097] The results showed that mannitol as a freeze-drying protectant had a protective effect on the purified paclitaxel albumin nanoparticles, and the addition of sucrose further ensured the freeze-drying stability of paclitaxel nanoparticles (the change in transmittance between 0 and 24 hours was small). The combination of mannitol / sucrose as a freeze-drying protectant had a good protective effect on both the dialysate and the freeze-dried product.

[0098] After accelerated exposure and reconstitution, the particle sizes of freeze-dried powders 3-1, 3-2, 3-4, and 3-5 increased to different degrees, while the particle size and transmittance of freeze-dried powder 3-3 remained unchanged. As a freeze-dried protective agent, 5% mannitol + 1% sucrose was shown to be the most effective, and was satisfactory without the need for a separate osmotic pressure regulator.

[0099] In addition, freeze-dried powders 3-4 and 3-5 had almost constant particle size and transmittance, similar to freeze-dried powder 3-3, and 1% mannitol + 1% sucrose and 4% mannitol + 2% sucrose were the most effective freeze-dried protective agents.

[0100] Example 4 Preparation of freeze-dried powder of fast-dissolving nanoparticles 50g of paclitaxel was dissolved in 500ml of chloroform / ethanol (11:1, v / v) to prepare the oil phase solution, 2.08kg of human serum albumin solution (20% w / v) was weighed out and water was added up to 10L to prepare the aqueous phase solution, and the oil phase and aqueous phase were emulsified online using a high shear disperser to form a primary emulsion. Homogenization was performed using a high pressure homogenizer at 10000-20000psi to obtain a nanoemulsion, which was transferred to a falling film evaporator, set at a heating temperature of 50℃, and reduced pressure evaporation was performed at a vacuum of less than 80mbar to remove the organic solvent in the solution. The resulting paclitaxel-albumin nanoparticles had an average diameter of 118 and were a translucent suspension.

[0101] An appropriate amount of sodium chloride was added to the obtained suspension to make it 0.9%, and it was dialyzed using a 300 kD ultrafiltration membrane and then dialyzed against 4 times the volume of 5% mannitol + 1% sucrose. After dialysis, the average diameter of the paclitaxel albumin nanoparticles was 121 nm, and purified nanoparticles 4 were obtained as a white translucent liquid.

[0102] The purified nanoparticle suspension after dialysis was successfully filtered through a 0.22 micron sterilization cartridge (Sartorius, Germany), and there was no significant change in particle size after filtration, and no significant change was observed even when the suspension was left at room temperature for 48 hours. The suspension was freeze-dried in a freeze dryer for 80 hours to obtain a stable white cake, which was named freeze-dried powder 4 of fast-dissolving nanoparticles (abbreviated as freeze-dried powder 4).

[0103] The white cake obtained by freeze-drying was added with water for injection to prepare an isotonic solution, and the solution was shaken gently to completely dissolve the white cake. The white cake was completely dissolved in about 1 minute, and there was only slight foaming. After resuspension, no significant changes were observed even when the solution was left at room temperature for 48 hours.

[0104] Example 5 Preparation of further batches of freeze-dried powder of fast-dissolving nanoparticles Three batches of lyophilized powders of fast-dissolving nanoparticles, designated as 5-1, 5-2, and 5-3, respectively, were prepared using the method of Example 4. The sample names and main steps are shown in the table below. [Table 5]

[0105] Example 6 Protein to Drug Ratio Screening The purified nanoparticle suspension 3-3 obtained in Example 3 was dialyzed at different times, and the contents of human serum albumin and paclitaxel were measured after each dialysis according to "Detection Method (I) Measurement Method of Content of Each Component in the Product" to obtain the protein and drug ratio. The products after each dialysis were freeze-dried, and then 20ml of injection water was added and gently shaken to completely dissolve. The re-dissolution dispersion time and foaming of the freeze-dried samples after dialysis at different times were observed, and the following results were obtained.

[0106] [Table 6]

[0107] The results show that dialysis can effectively reduce the protein to drug ratio in purified nanoparticle suspension. As the dialysis rate increases, the redissolution time of the product is significantly shortened and the foaming situation is significantly improved. Combined with Example 3, when the albumin / paclitaxel ratio is less than 3, the dispersion time of the product is about 1 minute and foaming is significantly reduced. By continuing to increase the dialysis rate, the protein to drug ratio is further reduced, but the improvement effect on dispersion time and foaming is not so significant.

[0108] Example 7: Stability test under high temperature conditions The taxane albumin nanoparticle suspension (undialyzed) obtained by the method described in the first paragraph of Example 4 was freeze-dried, and the freeze-dried powder 4 of fast-dissolving nanoparticles was left under accelerated conditions at 40°C and 60°C for 5 and 10 days, respectively, and then removed and redissolved in 0.9% sodium chloride solution and 20 ml of injection water, respectively. The changes in dispersion time, particle size, and transmittance were recorded, and the results are shown as follows:

[0109] [Table 7]

[0110] The above results show that compared with the paclitaxel albumin nanoparticle freeze-dried powder, the fast-dissolving nanoparticle freeze-dried powder 4 showed no significant differences in particle size after redissolution under isotonic conditions or in transmittance after 24 hours of exposure under accelerated exposure under the test conditions, and was not inferior in stability; however, the redissolution rate was significantly faster and foaming was clearly reduced.

[0111] Example 8 Comparison of redissolution phenomenon The freeze-dried powder 4 of the fast-dissolving nanoparticles of Example 4 was added with 20 ml of injection water and shaken gently to completely dissolve. The white cake was completely dissolved in 24 seconds with only slight foaming.

[0112] Two commercially available Abraxane freeze-dried powder injections were taken, one of which was dispersed by adding 20ml of 0.9% sodium chloride solution according to the standard procedure in the specification, and it took 10 minutes for the sample to completely dissolve, and there was slight foaming. The other was added with 0.9% sodium chloride, but the injection angle and speed were not controlled, but even after 23 minutes the dispersion was insufficient, white solids were observed, and a lot of foaming occurred.

[0113] The nanoparticle lyophilized powder 4 and the resuspension of Abraxane demonstrated room temperature stability with no significant changes within 72 h.

[0114] As a result, it was found that Abraxane requires a complicated procedure for preparing the liquid before use, and if the procedure is inappropriate, problems such as excessive foaming and incomplete dissolution are likely to occur. The particle dispersion of the present invention does not require complicated procedures, has a dispersion time of less than 1 minute, which is significantly shorter than that of Abraxane, and causes significantly less foaming.

[0115] Example 9 Comparative quality study The freeze-dried powders of the fast-dissolving nanoparticles 5-1 to 5-3 of Example 5 were compared with commercially available Abraxane in terms of properties, basicity, moisture, dispersion time, average particle size, total impurities, human serum albumin multimers, paclitaxel binding rate, in vitro release rate, and content.

[0116] [Table 8]

[0117] The results showed that the composition of the present invention had significantly reduced human albumin content and reconstitution time, lower total impurity and human albumin multimer contents than Abraxane, and was substantially consistent in other indicators.

[0118] Example 10 Comparative study of high temperature stability The freeze-dried powder 5-1 of the fast-dissolving nanoparticles of Example 5 was placed under a condition of 60°C and compared with commercially available Abraxane in terms of properties, basicity, moisture, dispersion time, average particle size, participating substances, human serum albumin, human serum albumin multimer, paclitaxel binding rate, in vitro release rate and content, etc.

[0119] [Table 9]

[0120] The results showed that when left for 10 days under high temperature conditions, the freeze-dried powder of the fast-dissolving nanoparticles of the present invention had an increased total impurity level similar to that of Abraxane, and the human albumin content in Abraxane was decreased, whereas the freeze-dried powder of the fast-dissolving nanoparticles of the present invention had no significant change in its content. It was found that the freeze-dried powder of human albumin, which is the fast-dissolving nanoparticles of the present invention, is more stable than Abraxane under high temperature conditions.

[0121] Example 11 Comparative photostability study The freeze-dried powder 5-1 of the fast-dissolving nanoparticles of Example 5 was placed under light irradiation conditions and compared with commercially available Abraxane in terms of properties, basicity, moisture, dispersion time, average particle size, participating substances, human serum albumin, human serum albumin multimer, paclitaxel binding rate, in vitro release rate and content, etc.

[0122] [Table 10]

[0123] As a result, after being left under light irradiation conditions for 10 days, the total impurities in the freeze-dried powder of the rapidly dissolving nanoparticles of the present invention did not increase significantly compared to Abraxane, and no significant trends in changes were observed in other indicators.

[0124] Example 12 Measurement of the content of each component in the freeze-dried powder of fast-dissolving nanoparticles For the freeze-dried powder of the fast-dissolving nanoparticles in Examples 4-5, the contents of mannitol, sucrose, and sodium ions were measured according to "Detection Method (1) Measurement method for the content of each component in the product".

[0125] [Table 11]

[0126] Example 13 Pharmacokinetic studies in dogs The test drugs used were the commercially available formulation Abraxane and the lyophilized powder 4 of Example 4, and a pharmacokinetic study of both drugs was performed using beagle dogs. The dose was 5 mg / kg, and the administration time was controlled at 30 minutes. 2 ml of blood was collected from the tip of the dog's forelimb vein before administration (0 hours), 15 minutes during the infusion (0.25 hours from the start of administration), when the needle was removed (0.5 hours from the start of administration), and 0.58, 0.75, 1.0, 1.5, 2.5, 3.5, 4.5, 6.5, 8.5, and 24.5 hours from the start of administration, and placed in a heparin anticoagulant tube, shaken, centrifuged at 3000 rpm for 10 minutes, and plasma was separated. The plasma paclitaxel concentration was measured by HPLC / MS / MS, pharmacokinetic parameters were obtained, and drug concentration-time curves were plotted (Figures 1-2, main pharmacokinetic parameters are shown in Tables 12-13). Drug concentration-time curves for both formulations showed nearly perfect agreement between their in vivo behaviors.

[0127] Example 14 Allergic phenomena in pharmacokinetic studies in dogs In the pharmacokinetic study of dogs carried out in Example 13, side effects of both formulations on dogs during administration were observed. In the Abraxane group, all beagle dogs (100%) of the subjects experienced clear allergic reactions of various degrees, mainly violent writhing during administration, excessive salivation, and perioral skin erythema, and vomiting and urinary incontinence were observed during administration of individual experimental animals. On the other hand, in the fast-dissolving nanoparticle freeze-dried powder group 4, only some animals (50%) experienced mild writhing, salivation, and perioral skin erythema, and the symptoms were mild. It was found that the product of the present invention can significantly reduce the allergic reaction of drugs.

[0128] Example 15 Tissue Distribution The test drugs were selected from the commercially available formulation Abraxane and the freeze-dried powder 4 of the fast-dissolving nanoparticles of Example 4. 48 mice bearing human breast cancer JIMT-1 were randomly divided into two test groups, with six mice in each test group at each time point. The test mice were injected with both formulations at a dose of 20 mg / kg via the tail vein, and blood was collected 0.25, 1, 3, and 24 h after administration, and the mice were immediately killed. The tissues of the tumor, heart, liver, spleen, lung, kidney, stomach, small intestine, pancreas, and ovary were dissected, and after pretreatment such as homogenization, the drug concentration in each tissue was measured by protein precipitation-LC / MS / MS method. The main pharmacokinetic parameters were calculated using the non-arterial model of Phoenix WinNonlin 8.0 software (Pharsight, USA), and the results of tissue distribution are shown in Figure 3.

[0129] The results showed that paclitaxel was widely distributed in the mouse body, and was detected in tumor, liver, lung, heart, stomach, small intestine, spleen, and kidney tissues, with the highest exposure in the liver. Based on the in vivo drug exposure, the exposure of the two formulations in various tissues in the mouse body was basically the same, and no significant difference was observed between them.

[0130] Example 16 Inhibitory effect on human breast cancer JIMT-1 tumor Test model: Using a NU / NU nude mouse model bearing human breast cancer JIMT-1, the inhibitory effect of purified lyophilized powder 4 on human breast cancer JIMT-1 was examined and compared with that of the same dose of Abraxane.

[0131] Test method: Seventy NU / NU female mice were subcutaneously inoculated with human breast cancer JIMT-1 cells (1 × 10 7 / 0.1 mL / animal), 10 days after seeding, approximately 165 mm 3 40 animals with good tumor growth were selected and divided into 5 groups (Day 0) to balance the tumor volume, with 8 animals in each group, divided into a vehicle control group, 10, 20, and 40 mg / kg groups of lyophilized powder 4, and a 20 mg / kg group of Abraxane®. The animals were administered once a week, three times a week (abbreviated as qw×3, i.e., administration on D0, D7, and D14), and the test was terminated on Day 24.

[0132] Data processing was performed using SPSS 19.0 statistical software Repeated Measure analysis, with tumor volumes measured multiple times over time.

[0133] The results showed that 1) animals in each treatment group were well tolerated after intravenous administration qw×3 in JIMT-1 tumor-bearing mice. 2) Under the test conditions, lyophilized powder 4 significantly inhibited the growth of human breast cancer JIMT-1 xenograft tumors in a dose-dependent manner at 10, 20, and 40 mg / kg intravenous administration compared to the vehicle control group. 3) At the equivalent dose (20 mg / kg), tumor inhibition by lyophilized powder 4 was equivalent to that of Abraxane. Tumor growth curves for each group are shown in Figure 4.

[0134] Example 17 Inhibitory effect on human breast cancer KPL-4 tumor Experimental model: Using a NU / NU nude mouse model bearing human breast cancer KPL-4, the inhibitory effect of purified lyophilized powder 4 on human breast cancer KPL-4 was investigated and compared with that of the same dose of Abraxane.

[0135] Test method: Seventy NU / NU female mice were subcutaneously inoculated with human breast cancer KPL-4 cells (1 × 10 7 / 0.1 mL / animal), 12 days after seeding, approximately 105 mm 3 48 animals with good tumor growth were selected and divided into 6 groups (Day 0) to balance the tumor volumes: a vehicle control group, 15, 30, and 60 mg / kg groups of lyophilized powder 4, and 30 and 60 mg / kg groups of Abraxane (registered trademark). The administration frequency was qw×3, and the study was terminated on D22.

[0136] Data processing was performed using SPSS 19.0 statistical software Repeated Measure analysis, with tumor volumes measured multiple times over time.

[0137] The results showed that: 1) under the test conditions, intravenous administration of 15, 30, and 60 mg / kg of lyophilized powder 4 significantly inhibited the growth of human breast cancer KPL-4 xenograft tumors in a dose-dependent manner compared to the vehicle control group. 2) At the same doses (30 mg / kg and 60 mg / kg), the tumor inhibition by lyophilized powder 4 was equivalent to that of Abraxane. The tumor growth curves for each group are shown in Figure 5.

[0138] Example 18 Inhibitory effect on human pancreatic cancer CFPAC-1 tumor Experimental model: Using a NU / NU nude mouse model bearing human pancreatic cancer CFPAC-1, the inhibitory effect of purified lyophilized powder 4 on human pancreatic cancer CFPAC-1 was investigated and compared with that of the same dose of Abraxane.

[0139] Test method: Seventy NU / NU female mice were subcutaneously inoculated with human pancreatic cancer CFPAC-1 cells (1 × 10 7 / 0.1 mL / animal), 7 days after seeding, approximately 195 mm 3Thirty-five animals with good tumor growth were selected and divided into five groups (Day 0) to balance the tumor volumes: a vehicle control group, 10, 20, and 40 mg / kg groups of lyophilized powder 4, and a 20 mg / kg group of Abraxane (registered trademark). The administration frequency was qw×3 for all groups, and the study was terminated on D23.

[0140] Data processing was performed using SPSS 19.0 statistical software Repeated Measure analysis, with tumor volumes measured multiple times over time.

[0141] The results showed that 1) after intravenous administration qw×3, each treatment group was well tolerated in CFPAC-1 tumor-bearing mice. 2) Under the test conditions, lyophilized powder 4 significantly inhibited the growth of human pancreatic cancer CFPAC-1 tumors in a dose-dependent manner at 10, 20, and 40 mg / kg intravenous administration compared to the vehicle control group. 3) At the equivalent dose (20 mg / kg), tumor inhibition by lyophilized powder 4 was equivalent to that of Abraxane. Tumor growth curves for each group are shown in Figure 6.

[0142] Example 19 Toxicity test of repeated intravenous administration for 3 weeks in Beagle dogs Forty-two Beagle dogs, half male and half female, were randomly divided into seven groups: low dose of lyophilized powder 4 (0.3 mg / kg), medium dose of lyophilized powder 4 (0.6 mg / kg), high dose of lyophilized powder 4 (1.2 mg / kg), Abraxane® (1.2 mg / kg), negative control (0.9% sodium chloride injection), vehicle control group-1 (1.7 mg / kg human serum albumin, which has the same protein content as the high dose of lyophilized powder 4), and vehicle control group-2 (10.8 mg / kg human serum albumin, which has the same protein content as the Abraxane® group). Intravenous bolus injections were performed once a week for a total of three times, with different volumes at equal concentrations (5 mg / ml).

[0143] The results showed that no animals died or became moribund during the study, and no abnormalities were observed in body weight, food intake, body temperature, hematology and coagulation, serum biochemistry results, urine, fecal occult blood, or anatomical findings.

[0144] In the vehicle control group 2, 2 / 3 female animals vomited yellow foam after D15 administration, and in the commercial control group, 1 / 3 male animals showed allergic reactions such as inability to stand and decreased activity (which recovered after intramuscular administration of dexamethasone). No allergic reactions were observed in the remaining dose groups.

[0145] At equal doses, the body exposure of paclitaxel (C max and AUC (0-t) ) was not statistically significant.

[0146] Conclusion: Under the experimental conditions, the in vivo PK properties of lyophilized powder 4 and Abraxane® are similar. Lyophilized powder 4 significantly reduced allergic reactions.

[0147] Example 20 In vitro hemolysis test An in vitro hemolysis test was performed using New Zealand white rabbit red blood cells, and samples were added using lyophilized powder 4 at a concentration of 5 mg / mL. A negative control group (0.9% sodium chloride injection), an excipient control group (human serum albumin) and a positive control group (deionized water) were also set up.

[0148] The results showed that no hemolysis or red blood cell agglutination occurred in the negative control tube (0.9% sodium chloride injection), while hemolysis occurred in the positive control tube (deionized water), proving that this test system accurately reflects the hemolytic effect of red blood cells caused by the sample / control.No hemolysis or red blood cell agglutination occurred in the sample tubes and vehicle control tubes during the test.

[0149] As a result, no hemolysis or red blood cell aggregation occurred with freeze-dried powder 4 at a concentration of 5 mg / mL.

[0150] Example 21 Vascular Muscle Stimulation Test For this study, nine New Zealand white rabbits were randomly divided into three groups, regardless of sex: vehicle control group (human serum albumin dose of 22.8 mg / kg, which is the same protein content as the high dose group), lyophilized powder 4 at 11 mg / kg, and 16.5 mg / kg. Intravenous bolus administration was performed twice a week in different volumes at equal concentrations (5 mg / ml). On the final administration day of the vascular stimulation test, lyophilized powder 4 was administered intramuscularly to the hind limbs, and the experiment used left and right identical controls, which were injected intravenously or intramuscularly with 0.9% sodium chloride injection.

[0151] As a result, macroscopic and histopathological examinations of all animals revealed no specimen-related vascular or muscle irritation.

[0152] Example 22 Acute toxicity test in CD1 mice In this study, 80 CD1 mice, half male and half female, were randomly divided into 8 groups: negative control (0.9% sodium chloride injection), vehicle control (103.5 mg / kg human serum albumin, which has the same protein content as the high dose group), lyophilized powder 4 at 25 mg / kg, 75 mg / kg, and 225 mg / kg, and commercial control Abraxane® at 25 mg / kg, 75 mg / kg, and 225 mg / kg. Three intravenous bolus doses of equal concentration (5 mg / ml) and different volumes were administered, with at least 4 hours between doses.

[0153] As a result, no animal deaths / moribunds were observed in any of the groups. The toxicity profile of Lyophilized Powder 4 and Abraxane® was similar, with a maximum tolerated dose (MTD) of 225 mg / kg / day.

[0154] Example 23 Preparation of fast-dissolving nanoparticles of docetaxel albumin and subsequent characterization 3 g of docetaxel was dissolved in 15 ml of dichloromethane / ethanol (1:1, v / v), and 500 ml of human serum albumin solution (4% w / v) was added. The mixture was homogenized using a high shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion, and then homogenized using a high pressure homogenizer at 10,000-20,000 psi to obtain a nanoemulsion. The nanoemulsion was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting docetaxel albumin nanoparticles had an average diameter of 102 nm and were a translucent suspension.

[0155] The obtained docetaxel albumin nanoparticle suspension was dialyzed using a 100 kD ultrafiltration membrane and 0.9% sodium chloride solution. The average diameter of the nanoparticles after dialysis at 5 times the volume was 104 nm in all cases, and there was no significant change in particle size during the dialysis process. A docetaxel purified nanoparticle suspension was obtained as a white translucent liquid.

[0156] The docetaxel nanoparticle suspension after dialysis was passed through a 0.22 micron filter, and the docetaxel content in the solution was measured by HPLC. The docetaxel content was adjusted to 50 mg / bottle according to the content, and the suspension was aliquoted and freeze-dried in a freeze dryer for 75 hours to obtain a stable white cake.

[0157] The white cake obtained by lyophilization was added with injection water and gently shaken until completely dissolved. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and docetaxel in the product were measured, and the ratio of albumin to docetaxel was 0.83:1.

[0158] Example 24 Preparation of fast-dissolving nanoparticles of cabazitaxel albumin and subsequent characterization 500mg of cabazitaxel was dissolved in 3ml of chloroform / ethanol (8:1, v / v), 150ml of human serum albumin solution (5% w / v) was added, and homogenized with a high shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized at 10,000-20,000psi using a high pressure homogenizer to obtain a nanoemulsion, which was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40mbar to remove the organic solvent in the solution. The resulting cabazitaxel albumin nanoparticles had a diameter of 100nm and were a translucent suspension.

[0159] An appropriate amount of sodium chloride was added to the obtained cabazitaxel albumin nanoparticle suspension to make the concentration 0.4%, and then dialyzed using a 100 kD ultrafiltration membrane and a 5% mannitol + 1% sucrose solution. When dialyzed at three times the volume, the average diameter of the nanoparticles was 101 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified cabazitaxel nanoparticle suspension was obtained, which was a white translucent liquid.

[0160] The purified nanoparticle suspension of cabazitaxel after dialysis was passed through a 0.22 micron filter, and the cabazitaxel content in the solution was measured by HPLC. The content was adjusted to 5 mg / bottle according to the content, and then dispensed and freeze-dried in a freeze dryer for 48 hours to obtain a stable white cake.

[0161] The white cake obtained by lyophilization was added with water for injection and gently shaken until completely dissolved. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and cabazitaxel in the product were measured, and the ratio of albumin to cabazitaxel was 2.53:1.

[0162] Example 25 Preparation of fast-dissolving nanoparticles of acetylated albumin of docetaxel and subsequent characterization 200mg of docetaxel acetyl derivative was dissolved in 2ml of chloroform / ethanol (1:1, v / v), 100ml of human serum albumin solution (5% w / v) was added, and homogenized with a high shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized at 10,000-20,000psi using a high pressure homogenizer to obtain a nanoemulsion, which was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40mbar to remove the organic solvent in the solution. The nanoparticle diameter of the docetaxel acetyl derivative albumin was 122nm, and the suspension was translucent.

[0163] The obtained nanoparticle suspension of docetaxel acetyl derivative albumin was dialyzed using a 1000 kD ultrafiltration membrane and a 5% mannitol + 0.2% sodium chloride solution. When dialyzed at four times the volume, the average diameter of the nanoparticles was 161 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified nanoparticle suspension of docetaxel acetyl derivative was obtained as a white translucent liquid.

[0164] The purified nanoparticle suspension of acetyl derivative of docetaxel after dialysis was passed through a 0.22 micron filter, and the content of acetyl derivative of docetaxel in the solution was measured by HPLC. The content was adjusted to 5 mg / bottle according to the content, and the suspension was aliquoted and freeze-dried in a freeze dryer for 48 hours to obtain a stable white cake.

[0165] The white cake obtained by lyophilization was added with injection water and gently shaken to completely dissolve. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and acetyl derivative of docetaxel in the product were measured, and the ratio of albumin to acetyl derivative of docetaxel was 1.66:1.

[0166] Example 26 Preparation of Rapidly Dissolving Nanoparticles of Rapamycin Albumin and Subsequent Characterization 166 mg of rapamycin was dissolved in 1 ml of chloroform, and 40 ml of human serum albumin solution (5% w / v) was added. The mixture was homogenized with a high-shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized at 10,000-20,000 psi using a high-pressure homogenizer to obtain a nanoemulsion. The mixture was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting rapamycin albumin nanoparticles had a diameter of 78 nm and were a translucent suspension.

[0167] An appropriate amount of sodium chloride was added to the obtained rapamycin albumin nanoparticle suspension to make the concentration 0.9%, and then dialysis was performed using a 100 kD ultrafiltration membrane and a 4% mannitol + 2% sucrose solution at a four-fold volume. The average diameter of the nanoparticles was 61 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified rapamycin nanoparticle suspension was obtained as a white translucent liquid.

[0168] The purified nanoparticle suspension of rapamycin after dialysis was passed through a 0.22 micron filter, and the rapamycin content in the solution was measured by HPLC. The content was adjusted to 10 mg / bottle according to the content, and then dispensed and freeze-dried in a freeze dryer for 75 hours to obtain a stable white cake.

[0169] The white cake obtained by lyophilization was added with water for injection and gently shaken until completely dissolved. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and rapamycin in the product were measured, and the ratio of albumin to rapamycin was 1.53:1.

[0170] Example 27 Preparation of fast-dissolving nanoparticles of temsirolimus albumin and subsequent characterization 101 mg of temsirolimus was dissolved in 0.5 ml of dichloromethane, and 19.5 ml of human serum albumin solution (5% w / v) was added. The mixture was homogenized for 2 minutes using a high-shear disperser (FLUKO FZ-20) to form a co-emulsion. The mixture was then homogenized at 10,000-20,000 psi using a high-pressure homogenizer to obtain a nanoemulsion. The nanoemulsion was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting temsirolimus albumin nanoparticles had a diameter of 88 nm and were a translucent suspension.

[0171] An appropriate amount of sodium chloride was added to the obtained temsirolimus albumin nanoparticle suspension to make the concentration 1.0%, and then dialyzed using a 100 kD ultrafiltration membrane and a 5% mannitol + 1% sucrose solution. When dialyzed at three times the volume, the average diameter of the nanoparticles was 77 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified temsirolimus nanoparticle suspension was obtained as a white translucent liquid.

[0172] The purified nanoparticle suspension of temsirolimus after dialysis was passed through a 0.22 micron filter, and the content of temsirolimus in the solution was measured by HPLC. The content was adjusted to 10 mg / bottle according to the content, and the suspension was aliquoted and freeze-dried in a freeze dryer for 66 hours to obtain a stable white cake.

[0173] 20 ml of water for injection was added to the white cake obtained by lyophilization and shaken gently to dissolve completely. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and temsirolimus in the product were measured, and the ratio of albumin to temsirolimus was 2.27:1.

[0174] Example 28 Preparation of fast-dissolving nanoparticles of epothilone B albumin and subsequent characterization 127 mg of epothilone B was dissolved in 2 ml of chloroform / ethanol (10:1, v / v), and 52 ml of human serum albumin solution (5% w / v) was added. The mixture was homogenized with a high-speed shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized with a high-pressure homogenizer at 10,000-20,000 psi to obtain a nanoemulsion, which was transferred to a rotary evaporator, heated in a 40°C water bath, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting epothilone B albumin nanoparticles had a diameter of 113 nm and were a translucent suspension.

[0175] The obtained nanoparticle suspension of epothilone B albumin was dialyzed using a 300 kD ultrafiltration membrane and a 5% mannitol + 0.5% sodium chloride solution. When dialyzed at 5 times the volume, the average diameter of the nanoparticles was 102 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified nanoparticle suspension of epothilone B was obtained as a white translucent liquid.

[0176] The purified nanoparticle suspension of epothilone B after dialysis was passed through a 0.22 micron filter, and the content of the acetyl derivative of epothilone B in the solution was measured by HPLC. The content was adjusted to 10 mg / bottle according to the content, and the suspension was aliquoted and freeze-dried in a freeze dryer for 65 hours to obtain a stable white cake.

[0177] 20 ml of water for injection was added to the white cake obtained by lyophilization and shaken gently to dissolve completely. The white cake was completely dissolved within 2 minutes, and the suspension was translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and epothilone B in the product were measured, and the ratio of albumin to epothilone B was 1.03:1.

[0178] Example 29 Preparation of fast dissolving nanoparticles of tanespimycin albumin and subsequent characterization 78 mg of tanespimycin was dissolved in 0.6 ml of chloroform, and 22 ml of human serum albumin solution (8% w / v) was added. The mixture was homogenized with a high-shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized at 10,000-20,000 psi using a high-pressure homogenizer to obtain a nanoemulsion. The nanoemulsion was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting tanespimycin albumin nanoparticles had a diameter of 105 nm and were a translucent suspension.

[0179] An appropriate amount of sodium chloride was added to the obtained tanespimycin albumin nanoparticle suspension to make it 0.9% concentration, and then dialyzed using a 300 kD ultrafiltration membrane and a 3% mannitol + 3% sucrose solution at a four-fold volume. The average diameter of the nanoparticles was 102 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified tanespimycin nanoparticle suspension was obtained, which was a purple liquid.

[0180] The purified nanoparticle suspension of tanespimycin after dialysis was passed through a 0.22 micron filter, and the tanespimycin content in the solution was measured by HPLC method. The content was adjusted to 10 mg / bottle according to the content, and then dispensed and freeze-dried in a freeze dryer for 45 hours to obtain a stable purple cake.

[0181] The purple cake obtained by lyophilization was added with injection water and gently shaken to completely dissolve. The purple cake was completely dissolved within 2 minutes, and the suspension was purple with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and tanespimycin in the product were measured, and the ratio of albumin to tanespimycin was 1.76:1.

[0182] Example 30 Preparation of fast-dissolving nanoparticles of 10-hydroxycamptothecin albumin and subsequent characterization 93 mg of 10-hydroxycamptothecin was dissolved in 2 ml of dichloromethane / ethanol (10:1, v / v), 48 ml of human serum albumin solution (5% w / v) was added, and the mixture was homogenized with a high-speed shear disperser (FLUKO FZ-20) for 2 minutes to form a co-emulsion. The mixture was then homogenized with a high-pressure homogenizer at 10,000-20,000 psi to obtain a nanoemulsion, which was transferred to a rotary evaporator, heated in a water bath at 40°C, and evaporated under reduced pressure at 40 mbar to remove the organic solvent in the solution. The resulting 10-hydroxycamptothecin albumin nanoparticles had a diameter of 118 nm and were a yellow translucent suspension.

[0183] The obtained 10-hydroxycamptothecin albumin nanoparticle suspension was dialyzed using a 300 kD ultrafiltration membrane and a 5% mannitol + 0.1% sodium chloride solution, and then dialyzed at four times the volume. The average diameter of the nanoparticles was 102 nm in all cases, and there was no significant change in particle size during the dialysis process. A purified 10-hydroxycamptothecin nanoparticle suspension was obtained as a yellow translucent liquid.

[0184] The purified nanoparticle suspension of 10-hydroxycamptothecin after dialysis was passed through a 0.22 micron filter, and the content of 10-hydroxycamptothecin in the solution was measured by HPLC method. The content was adjusted to 20 mg / bottle according to the content, and then dispensed and freeze-dried in a freeze dryer for 45 hours to obtain a stable yellow cake.

[0185] The yellow cake obtained by lyophilization was added with injection water and gently shaken until completely dissolved. The yellow cake was completely dissolved within 2 minutes, and the suspension was yellow and translucent with slight foaming. The particle size of the suspension after resuspension did not change significantly compared to before lyophilization. The properties and transmittance did not change significantly after being left at room temperature for 24 hours. The contents of human serum albumin and 10-hydroxycamptothecin in the product were measured, and the ratio of albumin to 10-hydroxycamptothecin was 1.49:1.

Claims

1. A fast-dissolving nanoparticulate composition comprising an active ingredient, a protein and a particle stabilizer, and optionally a lyoprotectant.

2. 10. The fast-dissolving nanoparticle composition of claim 1, comprising an active ingredient, a protein, a lyoprotectant and a particle stabilizer.

3. The rapidly soluble nanoparticle composition according to claim 1 or 2, wherein the active ingredient is selected from active ingredients suitable for encapsulation in human serum albumin and has the properties of being insoluble or poorly soluble in water and soluble or easily soluble in a specific organic solvent.

4. 4. The fast-dissolving nanoparticle composition of claim 3, wherein the active ingredient is selected from taxanes (including but not limited to paclitaxel, docetaxel, cabazitaxel, hydrophobic derivatives of docetaxel), macrolides (including but not limited to rapamycin and its derivatives, epothilone B and its derivatives, tanespimycin and its derivatives, etc.), camptothecins (including but not limited to 10-hydroxycamptothecin, SN-38 and its derivatives, etc.), anthracyclines (including but not limited to aclacinomycin and pirarubicin), or other active ingredients (including colchicine and its derivatives, thiocolchicine dimer, amiodarone, liothyronine, cyclosporine, exemestane, flutamide, fulvestrant, romidepsin, semustine, ibuprofen, etc.), preferably paclitaxel or docetaxel.

5. The fast-dissolving nanoparticle composition according to claim 1 or 2, wherein the protein is selected from serum albumin having a carrier effect, such as human serum albumin and bovine serum albumin, preferably human serum albumin.

6. 3. The fast dissolving nanoparticle composition according to claim 1 or 2, wherein the lyoprotectant is selected from mannitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, preferably a combination of mannitol and sucrose, the ratio of the two being preferably 10:1-1:1, preferably 8:1-3:1, most preferably 5:

1.

7. 3. The fast-dissolving nanoparticle composition according to claim 1 or 2, wherein the particle stabilizer is selected from sodium chloride, disodium hydrogen phosphate / sodium dihydrogen phosphate, potassium chloride.

8. The fast-dissolving nanoparticle composition according to claim 1 or 2, wherein the particle stabilizer is selected from sodium chloride.

9. The fast-dissolving nanoparticle composition according to claim 1 or 2, wherein each component in the fast-dissolving nanoparticle composition is, by weight, 1-30% active ingredient, 0.1-30% protein, 0.00001-3% particle stabilizer, and the remainder being a freeze-drying protection agent.

10. The fast-dissolving nanoparticle composition of claim 9, wherein the content of the particle stabilizer in the fast-dissolving nanoparticle composition is, in weight percent, 0.00001-3%, preferably 0.0001-1%, or any subrange therebetween, such as 0.0001-1%, 0.0001-0.1%, 0.0005-0.5%, 0.001-0.1%, 0.005-0.05%, 0.008-0.022%.

11. The fast-dissolving nanoparticle composition of claim 9, wherein the content of the active ingredient in the fast-dissolving nanoparticle composition is, by weight percent, 1-30%, preferably 2-20%, or any subrange of the ranges, such as 3-15%, 5-10%.

12. The fast-dissolving nanoparticle composition of claim 9, wherein the content of human serum albumin in the fast-dissolving nanoparticle composition is, by weight percent, 0.1-30%, or any subrange of the ranges of 1-25%, 5-20%.

13. The fast-dissolving nanoparticle composition according to claim 1 or 2, wherein the binding rate between the protein and the active ingredient is more than 90%, preferably ≧94%.

14. In the composition, the weight ratio of protein to active ingredient is 0.1:1-5:1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.70:1, 0.8:1, 0.9:1, 0.95:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1 3. The rapidly dissolving nanoparticle composition of claim 1, wherein the ratio is 1.8:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, or a range selected from between any two of the above.

15. In the composition, the average particle size of the fast-dissolving nanoparticles is 30-200 nm, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 198, 200, 201, 202, 203, 204, 205, 206, 207, 3. The rapidly dissolving nanoparticle composition of claim 1, wherein the nanoparticle size is 2, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 170, 175, 180, 185, 190, 195, 200 nm, or a range selected from between any two of the above numerical values.

16. The fast-dissolving nanoparticle composition of claim 1 or 2, wherein the composition does not contain any surfactant.

17. 3. A pharmaceutical composition comprising the rapidly dissolving nanoparticle composition of claim 1 or 2 in solid or liquid form.

18. 18. The pharmaceutical composition of claim 17, which is an injectable solution, a dry powder or a lyophilized powder.

19. 19. The pharmaceutical composition according to claim 18, wherein when the composition is provided in liquid form, the fast-dissolving nanoparticles are suspended in a pharma- ceutical acceptable carrier, including but not limited to buffers, preservatives, water for injection, saline, isotonic solutions, and the content of the active agent in the liquid form of the pharmaceutical composition is 0.1-100 mg / ml, preferably 0.5-50 mg / ml, more preferably 1-20 mg / ml, e.g. 5 mg / ml.

20. The pharmaceutical composition of claim 18, wherein the content of the active agent in the fast-dissolving nanoparticulate composition is 1-30% by weight, preferably 2-20% or 3-15% or 5-10%, when the composition is provided in solid form.

21. (1) a step of dissolving an active ingredient in an organic solvent to prepare an organic phase and dissolving a protein in water to prepare an aqueous phase; (2) a step of mixing and homogenizing the organic phase and the aqueous phase to form a nanoemulsion; (3) a step of removing the organic solvent in the nanoemulsion to obtain a suspension; and (4) a dialysis step, (5) Optionally, freeze-drying the mixture A method for producing the rapidly dissolving nanoparticulate composition according to any one of claims 1 to 16.

22. 22. The method of claim 21, wherein the organic solvent is selected from a low water solubility, low boiling point pure solvent, or a mixed solvent with a small molecule alcohol, preferably the organic solvent is selected from one or more of chloroform, dichloromethane, ethanol, or tert-butanol, preferably chloroform or a combination of chloroform and ethanol.

23. 22. The method of claim 21, wherein the dialysis method comprises adding a particle stabilizer to the suspension obtained in step (3) to make the concentration of the particle stabilizer in the suspension 0.01%-1.4% (w / v), and dialyzing with an aqueous solution of a lyoprotectant, preferably the concentration of the particle stabilizer in the suspension is 0.1-1.4% (w / v), preferably 0.1-0.9% (w / v), and preferably the aqueous solution of the lyoprotectant contains 1%-10%, preferably 2%-8%, preferably 5%-6% of the lyoprotectant.

24. 22. The method of claim 21, wherein the dialysis method is to perform dialysis with an aqueous solution of a lyoprotectant, the aqueous solution further comprising a particle stabilizer, the concentration of the particle stabilizer being 0.01%-1.4% (w / v), preferably 0.05%-0.9% (w / v), preferably 0.05%-0.5% (w / v), more preferably 0.05%-0.15%, preferably the aqueous solution of lyoprotectant comprises 1%-10%, preferably 2%-8%, preferably 5%-6% of the lyoprotectant.

25. 22. The method of claim 21, wherein the dialysis method is to perform dialysis with a particle stabilizer solution of an appropriate concentration, the particle stabilizer solution having a concentration of 0.01%-1.4% (w / v), preferably 0.1%-1.4% (w / v), more preferably 0.1%-0.9% (w / v), and optionally adding a lyoprotectant after completion of dialysis, preferably adding a lyoprotectant so that the resulting liquid contains 1%-10%, preferably 2%-8%, more preferably 5%-6%.

26. The method according to claim 21, wherein the freeze-drying conditions in step (4) are freezing at a temperature between -20°C and 60°C and drying at a temperature between 0°C and +40°C.

27. The method according to claim 21, wherein the dialysis step is performed at a ratio of 2 to 10, preferably 3 to 6.

28. 26. The method of any of claims 24-25, wherein the particle stabilizer is sodium chloride.

29. 29. A rapidly dissolving nanoparticulate composition comprising an active ingredient, a protein, a lyoprotectant and a particle stabilizer, produced by the process of any one of claims 21-28.

30. 30. Use of the rapidly dissolving nanoparticles according to any of claims 1-16 or 29 or the pharmaceutical composition according to any of claims 17-20 in the manufacture of a medicament for the treatment of a disease responsive to an active ingredient.

31. 31. The use according to claim 30, wherein the disease is selected from cancer, preferably from liver cancer, prostate cancer, lung cancer, breast cancer, multiple myeloma, transplant rejection, colon cancer, lymphoma.

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