Method for producing albumin-bound taxane nanoparticles with improved stability in maintaining particle size distribution

Abstract

The present invention relates to nanoparticles containing taxane and albumin and having improved stability in maintaining particle size distribution, and a method for producing the same. The method for producing nanoparticles of the present invention is useful as an improved method for producing nanoparticles since the average size of the nanoparticles is uniform over time and the structural stability and particle size distribution are excellent compared to nanoparticles containing taxane and albumin produced by conventional processes.

Description

[Technical Field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2021-0150107, filed with the Korean Intellectual Property Office on November 3, 2021, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method for producing albumin-bound taxane nanoparticles with improved stability in maintaining particle size distribution. [Background technology]

[0003] Poorly soluble drugs are drugs that are poorly soluble in water due to the hydrophobic moiety of their compound structure, and their practical use is often limited by poor solubility. For example, approximately 41% of new drugs under development are abandoned due to poor solubility, and approximately one-third of drugs listed in the US Pharmacopeia are classified as poorly soluble drugs. To use such poorly soluble drugs, additional substances must be added to overcome the poor solubility, but there have been many reported cases where the toxicity of the additional substances limits their use. For example, emulsification using emulsifiers and entrapment using liposomes are commonly used to solubilize poorly soluble substances in water, but these methods currently have limited use due to contamination with foreign substances not derived from the human body and physical instability.

[0004] Paclitaxel (PTX) is a diterpenoid derivative extracted from the bark of the yew tree (Taxus brevifolia). Its basic alkaloid structure consists of a taxane ring and an ester side chain. Paclitaxel is known to have anti-mitotic activity by promoting the transformation of tubulin complexes into stable microtubules, and is known to be effective against various cancers, including lung and breast cancer. While paclitaxel has shown great potential as an anticancer drug, its poor aqueous solubility has limited its widespread use in cancer treatment. To overcome its poor solubility, paclitaxel was previously dissolved in ethanol. Recently, however, paclitaxel has been conjugated to albumin for improved delivery efficiency and used as an injectable drug. Additionally, the aqueous solubility of paclitaxel has been improved using a solvent called Cremophor EL, a mixture of polyoxyethylated castor oil and absolute ethanol, for systemic administration. A commercial formulation in this form is Taxol™. However, clinical studies have shown that excessive administration of this solvent can cause side effects such as cardiotoxicity, neurotoxicity, neuropathy, and hypersensitivity.

[0005] Abraxane™ is a nanoparticle formulation of paclitaxel bound to human serum albumin to overcome the drawbacks of Taxol, such as the side effects caused by the solvent. It has demonstrated improved tumor responses in human and animal studies and has been used successfully as an anticancer therapeutic agent to date. However, the manufacturing method for Abraxane disclosed in prior patent document 1 (PCT / US1998 / 013272) requires rapid vacuum drying immediately after high-pressure homogenization of a paclitaxel and albumin mixture. If high-pressure homogenization is not immediately followed by rapid vacuum drying, the particle size of the nanoparticles containing paclitaxel and albumin increases significantly over time. If the particle size distribution of the nanoparticles after high-pressure homogenization does not remain uniform but increases rapidly, complex sequential processes must be adopted, which is extremely disadvantageous for scale-up. Furthermore, rapid vacuum drying is required, and an additional process must be performed, if necessary, to remove nanoparticles that have aggregated to an extent that they cannot be used as a drug. Furthermore, in the process disclosed in Patent Document 1, the concentrations of the starting materials, paclitaxel solution and albumin solution, are low, at 7.5% (w / v) and 3% (w / v), respectively, based on Example 5. If a scale-up facility is constructed using this process, the volume of the equipment, such as a mixing tank, must be increased. Furthermore, a concentration process must be performed to reduce the volume of the final dispersion, which results in a significant reduction in the amount of nanoparticles produced.

[0006] Numerous papers and patent documents are cited throughout this specification, and citations thereof are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art to which this invention pertains and the content of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have conducted extensive research to derive a method for producing albumin-bound taxane nanoparticles that can overcome the problems of Patent Document 1. As a result, they have confirmed that a method for producing nanoparticles comprising taxane and albumin according to one embodiment of the present invention overcomes the limitation of the process disclosed in Patent Document 1, namely, the problem of a sudden increase in nanoparticle size after high-pressure homogenization and before and / or during vacuum drying. Furthermore, they have demonstrated that the production method of the present invention can provide an environment suitable for scale-up in terms of economy and efficiency, and have thus completed the present invention.

[0008] It is therefore an object of the present invention to provide a method for producing nanoparticles comprising a taxane and albumin that have improved stability in maintaining the particle size distribution.

[0009] Another object of the present invention is to provide nanoparticles containing taxane and albumin produced by the above method.

[0010] It is yet another object of the present invention to provide a method for stabilizing and maintaining the particle size distribution of nanoparticles comprising a taxane and albumin.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims, and the drawings. [Means for solving the problem]

[0012] As used herein, the term "about" used in connection with numerical limitations, e.g., temperature, time, pressure, amount, concentration, etc., including ranges, can mean (+) or (-) 10%, 5%, 4%, 3%, 2%, 1%, or a range or specific value therebetween.

[0013] The term "nanoparticle" as used herein refers to particles having a particle size dimension of less than 1 μm. When the nanoparticles of the present invention are for intravenous injection, the nanoparticles preferably have a size of 500 nm or less. Specifically, the nanoparticles may have a size of 50 to 500 nm, 50 to 400 nm, 50 to 300 nm, 50 to 200 nm, 50 to 190 nm, 50 to 180 nm, 50 to 170 nm, 50 to 160 nm, 50 to 150 nm, 80 to 500 nm, 80 to 400 nm, 80 to 300 nm, 80 to 200 nm, 80 to 190 nm, 80 to 180 nm, 80 to 170 nm, 80 to 160 nm, 80 to 150 nm, 100 to 500 nm, It is preferable that the size is 100 to 400 nm, 100 to 300 nm, 100 to 200 nm, 100 to 190 nm, 100 to 180 nm, 100 to 170 nm, 100 to 160 nm, 100 to 150 nm, 110 to 500 nm, 110 to 400 nm, 110 to 300 nm, 110 to 200 nm, 110 to 190 nm, 110 to 180 nm, 110 to 170 nm, 110 to 160 nm, or 110 to 150 nm.

[0014] In a population of particles, the size of the population particles can be expressed as a mean and / or percentile. For example, D10 is the size below which 10% of the particles are smaller, D50 is the size below which 50% of the particles are smaller, and D90 is the size below which 90% of the particles are smaller. The average size of the particles may be expressed as D50.

[0015] In one embodiment of the present invention, the taxane concentration of the first solution may be 55-75% (w / v), 60-75% (w / v), 65-75% (w / v), 70-75% (w / v), 55-70% (w / v), 55-65% (w / v), 55-60% (w / v), 60-70% (w / v), 64-68% (w / v), or 65-67% (w / v).

[0016] In one embodiment of the present invention, the solvent of the first solution is selected from the group consisting of, but not limited to, ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone, and combinations thereof.

[0017] In a specific embodiment of the present invention, the solvent of the first solution is a mixed solvent of ethanol and chloroform, and the volume ratio of ethanol to chloroform is 1:5 to 1:20, 1:6 to 1:20, 1:7 to 1:20, 1:8 to 1:20, 1:9 to 1:20, 1:10 to 1:20, 1:12 to 1:20, 1:14 to 1:20, 1:15 to 1:20, 1:5 to 1:18, 1:6 to 1:18, 1:7 to 1:18, 1:8 to 1:18, 1:9 to 1:18, 1:10 to 1:18, 1:12 to 1:18, 1:14 to 1:18, 1:15 to 1: The ratio may be, but is not limited to, 1:18, 1:5 to 1:16, 1:6 to 1:16, 1:7 to 1:16, 1:8 to 1:16, 1:9 to 1:16, 1:10 to 1:16, 1:12 to 1:16, 1:14 to 1:16, 1:15 to 1:16, 1:5 to 1:15, 1:6 to 1:15, 1:7 to 1:15, 1:8 to 1:15, 1:9 to 1:15, 1:10 to 1:15, 1:12 to 1:15, or 1:14 to 1:15.

[0018] In one embodiment of the present invention, if the taxane concentration in the first solution is less than the lower limit of the range of 55 to 75% (w / v) defined in the present invention, even after dilution with water, the particle size may increase sharply to 200 nm or more before drying under reduced pressure, making the product unusable as an active ingredient in an injectable formulation.

[0019] Furthermore, if the taxane concentration in the first solution exceeds the upper limit of the range of 55 to 75% (w / v) defined in the present invention, paclitaxel will precipitate during the nanoparticle production process, making it impossible for the high-pressure homogenizer to operate normally and increasing the particle size of the nanoparticles.Therefore, if the concentration exceeds 75% (w / v), there is a problem that nanoparticles of uniform particle size cannot be produced.

[0020] In one embodiment of the present invention, the taxane may be, but is not limited to, paclitaxel, docetaxel, or a combination thereof.

[0021] In one embodiment of the present invention, the albumin concentration of the second solution may be 15-25% (w / v), 18-25% (w / v), 22-25% (w / v), 15-22% (w / v), 15-18% (w / v), 18-22% (w / v), or 19-21% (w / v).

[0022] In one embodiment of the present invention, the solvent of the second solution is an aqueous solvent selected from the group consisting of water, distilled water, sterile water, phosphate buffered saline (PBS), methanol, purified water, ethanol, 1-propanol, 2-propanol, 1-pentanol, 2-butoxyethanol, ethylene glycol, acetone, 2-butanone, 4-methyl-2-propanone, chloroform, and combinations thereof, but is not limited to this.

[0023] If the albumin concentration of the second solution is below the lower limit of the range of 15-25% (w / v) defined in the present invention, the volume of the dispersion must be excessively increased to achieve a paclitaxel:albumin weight ratio of 1:9 in the final nanoparticles, the same as that of Abraxane™, resulting in a volume outside the normal filling range and requiring an additional concentration process, making industrial application difficult. Furthermore, if the albumin concentration of the second solution exceeds the upper limit of the range of 15-25% (w / v) defined in the present invention, even after dilution with water, the average particle size will rapidly increase to 200 nm or more before drying under reduced pressure, making it unusable as an active ingredient in an injectable dosage form.

[0024] In one embodiment of the present invention, the albumin may be human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), or a combination thereof.

[0025] In one embodiment of the present invention, the volume ratio of the first solution to the second solution in step (c) is, but is not limited to, 1:20 to 1:45. For example, the volume ratio of the first solution to the second solution may be 1:20 to 1:43, 1:20 to 1:41, 1:20 to 1:39, 1:20 to 1:37, 1:20 to 1:35, 1:20 to 1:33, 1:20 to 1:31, 1:20 to 1:29, 1:20 to 1:27, 1:20 to 1:25, 1:20 to 1:23, 1:20 to 1:21, 1:22 to 1:45, 1:24 to 1:45, 1:2 The solubility may be 1:6 to 1:45, 1:28 to 1:45, 1:30 to 1:45, 1:32 to 1:45, 1:34 to 1:45, 1:36 to 1:45, 1:38 to 1:45, 1:40 to 1:45, 1:42 to 1:45, 1:44 to 1:45, 1:22 to 1:40, 1:25 to 1:40, 1:25 to 1:35, 1:25 to 1:30, 1:30 to 1:40, or 1:30 to 1:35.

[0026] A person skilled in the art can select an appropriate ratio to prepare the final nanoparticles with a paclitaxel:albumin weight ratio of about 1:9, the same as that of Abraxane™, and / or taking into consideration reactivity. The paclitaxel:albumin weight ratio is not necessarily limited to about 1:9, and may be in the range of 1:7-11, 1:8-10, or 1:8.5-9.5.

[0027] In one embodiment of the present invention, the preparation of the suspension in step (c) is carried out by high-pressure homogenization.

[0028] In one embodiment of the present invention, the high-pressure homogenization is performed at a temperature of, but not limited to, 10 to 40°C, 15 to 40°C, 20 to 40°C, 30 to 40°C, or 35 to 40°C.

[0029] In an embodiment of the present invention, the high pressure homogenization is carried out at a pressure of 10,000 to 30,000 psi, 12,000 to 30,000 psi, 15,000 to 30,000 psi, 16,000 to 30,000 psi, 17,000 to 30,000 psi, 18,000 to 30,000 psi, 10,000 to 25,000 psi, 12,000 to 25,000 psi, 15,000 to 25,000 psi, 16 The pressure may be, but is not limited to, 1,000 to 25,000 psi, 17,000 to 25,000 psi, 18,000 to 25,000 psi, 10,000 to 20,000 psi, 12,000 to 20,000 psi, 15,000 to 20,000 psi, 16,000 to 20,000 psi, 17,000 to 20,000 psi, or 18,000 to 20,000 psi.

[0030] The high pressure homogenization is carried out one or more times, more specifically, 1 to 5 times, 1 to 4 times, or 1 to 3 times, but is not limited thereto.

[0031] In one embodiment of the present invention, the dilution in step (d) may be performed at a volume of 3 to 12 times, 5 to 12 times, 7 to 12 times, 10 to 12 times, 3 to 10 times, 3 to 7 times, 3 to 5 times, 5 to 10 times, or 5 to 7 times the volume of the suspension, but is not limited thereto.

[0032] If the dilution ratio of the aqueous solvent is less than the lower limit of the range of 3 to 12 times defined in the present invention, even after the water dilution step, the particle size will increase sharply to 200 nm or more before drying under reduced pressure, making the nanoparticles unusable as an active ingredient in an injectable dosage form. This increase in particle size may be due to agglomeration between nanoparticles. Nanoparticle agglomeration can cause intravenous nanoparticles to clog blood vessels, so nanoparticle agglomeration and nanoparticle stability can affect the efficacy and safety of pharmaceutical compositions containing nanoparticles.

[0033] Furthermore, if the amount of aqueous solvent used exceeds the upper limit of the range of 3 to 12 times defined in the present invention, not only does the free fraction of paclitaxel, i.e., the unencapsulated rate, increase (unencapsulated rate of 0.72% when diluted 5.3 times with water to 6.24% when diluted 13 times with water), but also more water than necessary is used to stabilize the nanoparticle size, which results in an inefficient and uneconomical scale-up process.

[0034] In one embodiment of the present invention, the nanoparticles in the diluted solution in step (d) have an average particle size of, but not limited to, 100 to 200 nm, or 110 to 190 nm.

[0035] In an embodiment of the present invention, the method may further include (e) drying the diluted solution to obtain nanoparticles.

[0036] In an embodiment of the present invention, the drying may be, but is not limited to, vacuum drying, freeze drying, or heat drying.

[0037] When the drying according to an embodiment of the present invention is vacuum drying, it may be performed using a conventional vacuum drying method such as a rotary evaporator, and is not limited to a specific vacuum drying condition.

[0038] In specific embodiments of the present invention, the reduced pressure drying may be performed under temperature conditions in the range of, but not limited to, 10 to 70°C, 20 to 70°C, 30 to 70°C, 40 to 70°C, 10 to 60°C, 10 to 50°C, 10 to 40°C, 20 to 60°C, 30 to 50°C, 35 to 45°C, 38 to 42°C, or 39 to 41°C.

[0039] In specific embodiments of the present invention, the reduced pressure drying may be performed under a pressure condition in the range of, but not limited to, 10 to 110 hPa, 20 to 110 hPa, 30 to 110 hPa, 40 to 110 hPa, 50 to 110 hPa, 10 to 100 hPa, 10 to 90 hPa, 10 to 80 hPa, 10 to 70 hPa, 20 to 100 hPa, 30 to 90 hPa, 40 to 80 hPa, 50 to 70 hPa, or 55 to 65 hPa.

[0040] The nanoparticles present in the diluted solution can maintain an average particle size of 100 to 200 nm, preferably 110 to 190 nm, from before drying until drying is completed.

[0041] The method for producing nanoparticles comprising taxane and albumin provided in one aspect of the present invention exhibits remarkable advantages, such as the average size of the nanoparticles in the aqueous dilution solution remaining uniform over time before vacuum drying, and excellent structural stability and particle size distribution, compared to conventional processes that involve high-pressure homogenization and rapid vacuum drying without diluting with water, and these advantages are supported by the examples, comparative examples, and experimental examples described below.

[0042] According to another aspect of the present invention, there are provided nanoparticles containing a taxane and albumin, which are produced by the production method according to the above-mentioned aspect of the present invention.

[0043] In one embodiment of the present invention, the nanoparticles comprising taxane and albumin prepared by the manufacturing method according to one aspect of the present invention have substantially the same therapeutic activity and particle properties as the nanoparticles before drying (e.g., the nanoparticles in the diluent in step (d)) when the dried nanoparticles are reconstituted in an aqueous solvent (including in vivo and in vitro).

[0044] When the dried nanoparticles are reconstituted in an aqueous solvent, the average particle size and particle size distribution are substantially the same as those of the nanoparticles before drying. The term "substantially the same" means that the parameters of the nanoparticles immediately after freeze-drying are within a range of ±10%, more specifically, ±7%, ±5%, ±4%, or ±3.5%, compared to the parameters of the nanoparticles before freeze-drying.

[0045] In one embodiment of the present invention, the dried nanoparticles are stable at room temperature for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months, and retain the therapeutic activity and particle properties of the nanoparticles before drying when reconstituted in an aqueous solvent after storage at room temperature for such periods.

[0046] In a specific embodiment of the present invention, the drying is freeze-drying.

[0047] In one embodiment of the present invention, the freeze-drying refers to a process in which nanoparticles, which are the material to be dried, are frozen and then the frozen solvent is removed by sublimation in a vacuum environment. The freeze-drying process optionally includes the addition of an excipient or cryoprotectant to enhance the storage stability of the freeze-dried product.

[0048] The excipients or cryoprotectants include, but are not limited to, polymers such as dextran and polyethylene glycol; sugars such as sucrose, glucose, trehalose, and lactose; surfactants such as polysorbates; and amino acids such as glycine, arginine, and serine.

[0049] In one embodiment of the present invention, the nanoparticles may further comprise one or more additional therapeutic agents in addition to the taxane of the present invention.

[0050] In one embodiment of the present invention, the nanoparticles may be pharmaceutically formulated for administration to a patient. Various dosage forms and drug delivery systems are available in the art. See, for example, Gennaro, AR, ed. (1995) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.

[0051] In one embodiment of the present invention, the nanoparticles of the present invention may be administered as a pharmaceutical composition by any one of the following routes: orally; parenterally, for example, transdermally, intranasally, intramuscularly, intravenously, subcutaneously, intravascularly, intratumorally, etc.

[0052] In one embodiment of the present invention, the nanoparticles of the present invention may include a solid, liquid, semi-solid, or gaseous excipient such as:

[0053] The solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like.

[0054] The liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and petroleum, various oils including those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Preferred liquid carriers, particularly those for injectable solutions, include water, saline, aqueous dextrose, and glycols. Other suitable pharmaceutical excipients and their dosage forms are described in Remington's Pharmaceutical Sciences, edited by E.W. Martin (Mack Publishing Company, 18th ed., 1990).

[0055] In a specific embodiment of the present invention, the nanoparticles are formulated for intravenous injection. In another specific embodiment of the present invention, the nanoparticles are formulated for direct injection or perfusion into a tumor.

[0056] According to one aspect of the present invention, there is provided a method for stabilizing and maintaining the particle size distribution of nanoparticles, comprising the steps of: (a) preparing a first solution in which a taxane is dissolved at 55-75% (w / v); (b) preparing a second solution in which albumin is dissolved at 15-25% (w / v); (c) mixing the first solution and the second solution to form a suspension; and (d) diluting the suspension with an aqueous solvent in an amount 3 to 12 times the volume of the suspension.

[0057] The method for maintaining and stabilizing the particle size distribution of nanoparticles according to one embodiment of the present invention includes steps (a) to (d) identical to the method for producing nanoparticles according to one embodiment of the present invention, and therefore, the content common to both inventions will be omitted in this specification to avoid duplication. [Effects of the Invention]

[0058] The method for producing nanoparticles containing taxane and albumin of the present invention can be useful as an improved method for producing nanoparticles containing taxane and albumin, since the average size of the nanoparticles remains uniform over time and the structural stability and particle size distribution are excellent compared to nanoparticles produced by conventional processes. DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of more specifically illustrating the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0060] [Example] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise specified.

[0061] <Comparative Example 1> Preparation of nanoparticles containing paclitaxel and albumin by conventional method 1 Nanoparticles containing paclitaxel and albumin were prepared according to the method of Example 4 in Patent Document 1 (PCT / US1998 / 013272). Specifically, 30 mg of paclitaxel was dissolved in a chloroform / ethanol solvent (11:1 v / v) consisting of 0.55 ml of chloroform and 0.05 ml of ethanol to prepare a 5% (w / v) paclitaxel solution (first solution). Also, 29.4 ml of a 1% (w / v) human serum albumin (HSA) solution (second solution) pre-saturated with 1% chloroform was prepared. The first and second solutions were mixed and mixed in an IKA homogenizer (T-25) at 5,600 rpm for 5 minutes to form a coarse emulsion. The mixture was then transferred to a Micronox (MN400BF) and subjected to two high-pressure homogenization cycles at 22°C and 18,000 psi. After high-pressure homogenization, the mixture was transferred to a rotary evaporator and dried under reduced pressure at 40°C and 60 hPa for 15-30 minutes to rapidly remove the chloroform, producing nanoparticles containing paclitaxel and albumin.

[0062] <Comparative Example 2> Preparation of nanoparticles containing paclitaxel and albumin by conventional method 2 Nanoparticles containing paclitaxel and albumin were prepared according to the method of Example 5 in Patent Document 1 (PCT / US1998 / 013272). Specifically, 225 mg of paclitaxel was dissolved in a chloroform / ethanol solution (9:1 v / v) consisting of 2.7 ml of chloroform and 0.3 ml of ethanol to prepare a 7.5% (w / v) paclitaxel solution (solution 1). Furthermore, 97 ml of a 3% (w / v) human serum albumin (HSA) solution (solution 2) was prepared. The first and second solutions were mixed, and the mixture was homogenized in an IKA homogenizer (T-25) at 5,600 rpm for 5 minutes to form a coarse emulsion. The mixture was then transferred to a Micronox (MN400BF) and subjected to high-pressure homogenization at 22°C and 18,000 psi for two cycles. After the high-pressure homogenization, the mixture was transferred to a rotary evaporator and dried under reduced pressure at 40°C and 60 hPa for 15 to 30 minutes to quickly remove the chloroform, thereby producing nanoparticles containing paclitaxel and albumin.

[0063] <Examples 1 to 14> Preparation of nanoparticles containing paclitaxel and albumin according to the present invention The present inventors prepared nanoparticles containing paclitaxel and albumin as follows: The following experimental protocol corresponds to Example 3 in Table 1 below.

[0064] A 66.7% (w / v) paclitaxel solution (Solution 1) was prepared by dissolving 1 g of paclitaxel in 1.5 ml of a 14:1 volumetric chloroform / ethanol solvent (14:1 v / v). 45 ml of a 20% (w / v) albumin aqueous solution (Solution 2, Greencross Human Serum Albumin Inj. 20%) was also prepared. The first and second solutions were mixed and the mixture was homogenized at 5,600 rpm for 5 minutes using an IKA homogenizer (T-25). The mixture was then transferred to a Micronox (MN400BF) and subjected to two cycles of high-pressure homogenization at 22°C and 18,000 psi. The resulting mixture was diluted with 5.3 volumes of water. The diluted mixture was dried under reduced pressure at 40°C and 60 hPa using a rotary evaporator to produce nanoparticles containing paclitaxel and albumin.

[0065] Nanoparticles were prepared in the same manner as described above, except for varying the concentration of the first solution, the concentration of the second solution, or the amount of water used for dilution. Specific variable conditions for each example are shown in Table 1 below.

[0066] In Table 1 below, the volume ratio of the first and second solutions was determined based on the concentrations of the first and second solutions, so that the weight ratio of paclitaxel to albumin in the final nanoparticles would be 1:9, the same as that of Abraxane®.

[0067] [Table 1]

[0068] Comparing the manufacturing methods of Examples 1 to 14 listed in Table 1 above with the manufacturing methods (Comparative Examples 1 and 2) described in Examples 4 and 5 of Prior Patent Document 1 (PCT / US1998 / 013272), the differences are i) the concentrations of the first and second solutions, and ii) whether or not the mixed solution after high-pressure homogenization further includes a step of diluting it with water before the step of drying it under reduced pressure.

[0069] <Comparative Examples 3 to 12> Fabrication of nanoparticles containing paclitaxel and albumin without the dilution step The inventors conducted experiments in Comparative Examples 3 to 12 to confirm how the "step of diluting the mixed solution after high-pressure homogenization with water before the step of drying under reduced pressure," which is further included in the manufacturing methods of Examples 1 to 14 of the present invention compared to the manufacturing methods of Comparative Examples 1 and 2, affects the quality of the nanoparticles containing paclitaxel and albumin produced.

[0070] Specifically, nanoparticles containing paclitaxel and albumin were prepared using the same preparation method as in Examples 1 to 14, except that after mixing the first and second solutions and homogenizing them under high pressure, the water dilution step was not performed before drying under reduced pressure. Specific variable conditions for each comparative example are shown in Table 2 below.

[0071] [Table 2]

[0072] <Comparative Examples 13 to 24> i) First solution concentration (%(w / v)), ii) Second solution concentration (%(w / v)), and iii) Water volume used for dilution in the preparation of nanoparticles containing paclitaxel and albumin The inventors conducted experiments in Comparative Examples 13 to 24 to determine how i) the concentration of the first solution (% (w / v)), ii) the concentration of the second solution (% (w / v)), and iii) the amount of water used for dilution, which were set in Examples 1 to 14 of the present invention, affect the quality of the nanoparticles containing paclitaxel and albumin produced.

[0073] Specifically, nanoparticles containing paclitaxel and albumin were prepared by the same method as in Examples 1 to 14, except that the first solution concentration (% (w / v)), second solution concentration (% (w / v)), and water volume used for dilution were outside the ranges set in Examples 1 to 14. Specific variable conditions for each comparative example are shown in Table 3 below.

[0074] As in Examples 1 to 14 above, the volume ratio of the first and second solutions in the table below was determined based on the concentrations of the first and second solutions, so that the weight ratio of paclitaxel to albumin in the final nanoparticles would be 1:9, the same as that of Abraxane.

[0075] [Table 3]

[0076] <Experimental Example 1> Comparative evaluation of the superiority of particle size distribution maintenance of the nanoparticles of the present invention compared with the conventional process (PCT / US1998 / 013272) If the particle size distribution (PSD) of the nanoparticles in the high-pressure homogenized mixture is maintained constant until the solvent is completely removed in the vacuum drying step after high-pressure homogenization, nanoparticles containing paclitaxel and albumin can be produced at high productivity.

[0077] In addition, if the effect of maintaining a constant particle size distribution of nanoparticles even after a certain period of time has passed after high-pressure homogenization and before reduced-pressure drying is achieved, the stability of the particle size of nanoparticle products during the manufacturing process can be ensured, and the technology can be actively utilized as an economical, efficient, and scale-up-friendly technology.

[0078] Therefore, we compared and evaluated whether the particle size distribution of nanoparticles in the mixed liquid (water-diluted liquid in the examples) before drying under reduced pressure produced by the conventional manufacturing methods of Comparative Examples 1 and 2 and the manufacturing methods of Examples 1 to 14 of the present invention remains stable over time.

[0079] More specifically, a Zetasizer (Malvern Zetasizer, Malvern Instruments Ltd.) was used to compare and evaluate the particle size distribution of nanoparticles over time in the high-pressure homogenized mixtures in Comparative Examples 1 and 2 and in the diluted solutions diluted with water in Examples 1 to 14. The measurement method for the suspensions on the Zetasizer included adjusting the following parameters: temperature 25.0°C, scattering angle 90°, refractive index dispersion 1.33, and viscosity (automatic) 0.8872 cP. 1 ml of each redispersed suspension was diluted with 10 ml of triple-distilled water, and 1 ml of this was transferred to a cuvette and placed in the Zetasizer cuvette holder to begin particle size analysis. Multimode analysis was performed three times, and the average value was calculated.

[0080] The results are shown in Table 4 below.

[0081] [Table 4]

[0082] As can be seen from Table 4, the manufacturing method of the present invention (Examples 1 to 14), which includes a step of diluting with water after high-pressure homogenization and before vacuum drying, maintains the size of the nanoparticles for a long period of time, and has relatively excellent structural stability and particle size distribution, compared to the conventional manufacturing method (Comparative Examples 1 and 2), which does not include a dilution step with water and instead involves rapid vacuum drying after high-pressure homogenization.

[0083] Specifically, the nanoparticles of Comparative Examples 1 and 2, which correspond to Examples 4 and 5 of PCT / US1998 / 013272, were found to have Z-average (nm) values ​​of 430.1 nm and 458.6 nm or greater after 20 minutes of high-pressure homogenization, making them unusable as active ingredients in injectable dosage forms. In contrast, the nanoparticles of Examples 1 to 14 of the present invention all maintained Z-average (nm) values ​​of <200 nm even after 120 minutes, demonstrating excellent particle size distribution.

[0084] The fact that the nanoparticles in the mixed solution (aqueous dilution in the present embodiment) before drying under reduced pressure maintain an excellent particle size distribution is extremely advantageous in terms of configuring a scale-up process.

[0085] More specifically, when the particle size distribution of nanoparticles increases rapidly rather than remaining uniform after high-pressure homogenization, as in the process disclosed in PCT / US1998 / 013272, complex continuous processes must be adopted, making it extremely difficult to scale up, and vacuum drying must be performed quickly, and if necessary, an additional process must be performed to remove nanoparticles that have aggregated to the point where they are unusable as a drug. Furthermore, in the process disclosed in PCT / US1998 / 013272, the concentrations of the starting materials, paclitaxel solution and albumin solution, are low, at 7.5% (w / v) and 3% (w / v), respectively, based on Example 5. If scale-up equipment is constructed as is, the volume of equipment such as mixing tanks must be increased, and a concentration process must be performed to reduce the volume of the final dispersion, resulting in a significant decrease in nanoparticle production yield.

[0086] In contrast, when the particle size distribution of nanoparticles in the diluted solution is maintained uniformly through the dilution step, as in the method of <Examples 1-14> according to the present invention, there is no need to employ complex continuous processes, there is no need to rapidly perform vacuum drying, and there is no need for an additional process to remove nanoparticles that have aggregated to the extent that they cannot be used as a drug, thereby achieving a significant effect of significantly improving the final nanoparticle production yield. Furthermore, the manufacturing method provided by the present invention also achieves the effect of improving workability when considering scale-up, since the first and second solutions, which are raw materials, are used at high concentrations and the volume of the raw materials is small.

[0087] This shows that the method for producing nanoparticles provided in one aspect of the present invention not only solves the limitation of the process disclosed in PCT / US1998 / 013272, i.e., the problem of a sudden increase in the size of nanoparticles from high-pressure homogenization to reduced-pressure drying, but also provides an environment suitable for scale-up in terms of economy and efficiency.

[0088] <Experimental Example 2> Comparative evaluation of the superiority of particle size distribution maintenance of the nanoparticles of the present invention with and without the water dilution step The above-mentioned Examples 1 to 14 and Comparative Examples 3 to 12 differ in the process in whether or not a water dilution step is performed before vacuum drying after high-pressure homogenization, but the remaining conditions are all the same.

[0089] Using the same method as in Experimental Example 1, we compared and evaluated whether the particle size distribution of nanoparticles in the mixed liquid (water-diluted liquid in the examples) before drying under reduced pressure, produced in Examples 1 to 14 of the present invention and Comparative Examples 3 to 12, remained stable over time.

[0090] The results are shown in Table 5 below.

[0091] [Table 5]

[0092] As can be seen from Table 5, the nanoparticles in the diluted solutions of Examples 1 to 14 of the present invention, which were subjected to a water dilution step before vacuum drying after high-pressure homogenization, maintained a uniform particle size distribution over time. On the other hand, the nanoparticles in the mixed solutions of Comparative Examples 3 to 12, which did not include a water dilution step, rapidly increased in size over time to a level that made them unusable as active ingredients in injection dosage forms.

[0093] From the above results, it can be seen that the advantages described in Experimental Example 1 can be ensured when constructing a scale-up process only if the water dilution step included in the examples of the present invention is carried out.

[0094] <Experimental Example 3> Comparative evaluation of the excellence of particle size distribution maintenance of the nanoparticles of the present invention according to i) the concentration of the first solution (% (w / v)), ii) the concentration of the second solution (% (w / v)), and iii) the amount of water used for dilution In order to determine what problems may arise in the particle size distribution of the nanoparticles of the present invention depending on i) the concentration of the first solution (% (w / v)), ii) the concentration of the second solution (% (w / v)), and iii) the amount of water used for dilution, the inventors prepared nanoparticles according to the above-mentioned Comparative Examples 13 to 24 and measured the particle size distribution in the same manner as in Experimental Example 1.

[0095] (Experimental Example 3-1. Concentration of First Solution (% (w / v))) The manufacturing conditions for the above-mentioned Comparative Examples 13 to 16 are shown in Table 6, and the particle size distribution data is shown in Table 7.

[0096] [Table 6]

[0097] [Table 7]

[0098] -: Paclitaxel precipitated during the nanoparticle manufacturing process, making it impossible for the high-pressure homogenizer to operate normally, and the particle size of the nanoparticles increased to an unmeasurable level.

[0099] As can be seen from Table 7 above, when the concentration of the first solution was below the lower limit of the range of 55 to 75% (w / v) defined in the present invention (i.e., in the cases of Comparative Examples 13 and 14), even after the water dilution step, there was a problem in that the particle size suddenly increased to 200 nm or more before drying under reduced pressure, to an extent that it could not be used as an active ingredient in an injectable dosage form.

[0100] Furthermore, when the concentration of the first solution exceeded the upper limit of the range of 55-75% (w / v) defined in the present invention (i.e., in the cases of Comparative Examples 15 and 16), paclitaxel precipitated during the nanoparticle production process, making it impossible to operate the high-pressure homogenizer normally. Furthermore, the particle size of the nanoparticles also increased, and it was confirmed that nanoparticles with a uniform particle size could not be produced when the concentration of the first solution exceeded 75% (w / v).

[0101] (Experimental Example 3-2. Concentration of the second solution (% (w / v))) The manufacturing conditions for the above-mentioned Comparative Examples 17 to 20 are shown in Table 8, and the particle size distribution data is shown in Table 9.

[0102] [Table 8]

[0103] [Table 9]

[0104] As seen in Tables 8 and 9 above, when the concentration of the second solution is below the lower limit of the range of 15-25% (w / v) defined in the present invention (i.e., in the cases of Comparative Examples 17 and 18), the volume of the dispersion had to be excessively increased to ensure that the paclitaxel:albumin weight ratio in the final nanoparticles was 1:9, the same as that of Abraxane™, as shown in the mixed volume ratios of the first and second solutions of 1:120.0 and 1:60.0 in Table 8. As a result, the volume ratio of the mixed solution fell outside the normal filling volume range, requiring an additional concentration process, making industrial application difficult.

[0105] Furthermore, when the upper limit of the range of 15 to 25% (w / v) was exceeded (i.e., in the cases of Comparative Examples 19 and 20), even after dilution with water, the particle size rapidly increased to 200 nm or more before drying under reduced pressure, making it unusable as an active ingredient in an injection formulation.

[0106] (Experimental Example 3-3. Double water usage) The manufacturing conditions for the above-mentioned Comparative Examples 21 to 24 are shown in Table 10, and the particle size distribution data are shown in Table 11.

[0107] [Table 10]

[0108] [Table 11]

[0109] As can be seen from Table 11 above, when the amount of water used for dilution was less than the lower limit of the range of 3 to 12 times defined in the present invention (i.e., in the cases of Comparative Examples 21 and 22), even after the water dilution step, there was a problem in that the particle size suddenly increased to 200 nm or more before drying under reduced pressure, to an extent that the active ingredient could not be used as an injectable dosage form.

[0110] Furthermore, when the amount of water used exceeds the upper limit of the range of 3 to 12 times defined in the present invention (i.e., in the cases of Comparative Examples 23 and 24), not only does the free fraction of paclitaxel, i.e., the unencapsulated rate, increase (unencapsulated rate of 0.72% when diluted 5.3 times with water → unencapsulated rate of 6.24% when diluted 13 times with water), but also more water than necessary is used to stabilize the nanoparticle size, resulting in inefficient and uneconomical problems in the scale-up process.

[0111] The unencapsulated rate of paclitaxel was confirmed by the following method.

[0112] The samples prepared in Example 3 (5.3-fold dilution with water) or Comparative Example 23 (13-fold dilution with water) were ultrafiltered, and the lower layer was analyzed by HPLC.

[0113] <HPLC analysis of unencapsulated paclitaxel> Chromatography HPLC: Agilent Technologies G7111A solvent delivery system Agilent Technologies G7129A Auto Injector Agilent Technologies G7114A Variable Wavelength Detector Agilent Technologies G7116A Column Oven Column: FluoroSep-RP Phenyl, 5 μm, 4.6 mm x 25 cm, Perkin Elmer® Column temperature: 25℃ Mobile phase: water / acetonitrile 11:9 Flow rate: 1.5mL / min Detection: 227nm Injection volume: 10μL Analysis time: 20 minutes As a result, when diluted with 5.3 times the amount of water as in Example 3, the unencapsulated paclitaxel rate was 0.72%, and when diluted with 13 times the amount of water as in Comparative Example 23, the unencapsulated paclitaxel rate was 6.24%. The only difference in the manufacturing method between Example 3 and Comparative Example 23 is the water dilution rate, and therefore, it can be seen that the water dilution rate is an important factor in introducing a scale-up process to manufacture high-quality nanoparticles.

[0114] From the results of Experimental Examples 2 and 3 above, it can be seen that the nanoparticle manufacturing methods according to Examples 1 to 14 of the present invention typically organically link the concentrations of the first and second solutions and the step of diluting with water before drying under reduced pressure, thereby achieving the remarkable effect of maintaining a constant particle size distribution of the nanoparticles in the high-pressure homogenized diluted solution, and as a result, it is possible to manufacture nanoparticles containing paclitaxel and albumin at high productivity.

[0115] Furthermore, if the particle size distribution of nanoparticles remains constant even after a certain period of time after high-pressure homogenization, as in the present invention, the limitations of conventional processes, such as the disadvantages of scale-up, the need for complex continuous processes, and the need for an additional step to remove nanoparticles that have aggregated to the point where they cannot be used as drugs, are overcome, making this an economical and efficient technology that can be actively utilized as a technology suitable for scale-up.

[0116] <Experimental Example 4> Evaluation of particle size distribution after freeze-drying and reconstitution (uniformity of particle size and ease of reconstitution) After the process of preparing nanoparticles containing paclitaxel and albumin in Examples 1 to 14, it was subsequently confirmed whether the particle size remained uniform i) before freeze-drying the nanoparticles and ii) upon reconstitution after freeze-drying.

[0117] More specifically, the dispersion from which the organic solvent had been removed by drying under reduced pressure was filtered through a Sartorius filter with a 0.22 μm pore size. The filtrate was filled into a glass vial for lyophilization and freeze-dried for 72 hours. After freeze-drying, 20 ml of triple-distilled water or 0.9% saline was added to the vial containing the resulting freeze-dried powder, which was then redispersed. The particle size distribution measurement results are shown in Table 12 below.

[0118] [Table 12]

[0119] As can be seen from Table 12, it was confirmed that the nanoparticles prepared in Examples 1 to 14 according to the present invention maintained a uniform particle size before freeze-drying and when reconstituted after freeze-drying.

Claims

1. A method for producing nanoparticles comprising a taxane and albumin, comprising the steps of: (a) providing a first solution in which a taxane is dissolved at 55-75% (w / v); (b) preparing a second solution in which albumin is dissolved at 15-25% (w / v); (c) mixing the first solution and the second solution to form a suspension; and (d) diluting the suspension with an aqueous solvent in an amount 3 to 12 times the volume of the suspension.

2. The method of claim 1 , further comprising: (e) drying the diluted solution to obtain nanoparticles.

3. The method according to claim 2, wherein the nanoparticles present in the diluted solution maintain an average particle size of 100 to 200 nm from before drying to completion of drying.

4. The method of claim 2, wherein the nanoparticles present in the diluted solution maintain an average particle size of 110 to 190 nm from before drying to completion of drying.

5. The method of claim 1 , wherein the taxane is paclitaxel, docetaxel, or a combination thereof.

6. 2. The method of claim 1, wherein the albumin is human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), or a combination thereof.

7. 2. The method of claim 1, wherein in step (c), the volume ratio of the first solution to the second solution is 1:20 to 1:

45.

8. 2. The method of claim 1, wherein the suspension in step (c) is prepared by high-pressure homogenization.

9. The method according to claim 8, wherein the high-pressure homogenization is carried out at a temperature of 10 to 40°C.

10. The method according to claim 8, wherein the high-pressure homogenization is carried out under a pressure condition of 10,000 to 30,000 psi.

11. A method for stabilizing and maintaining the particle size distribution of nanoparticles comprising a taxane and albumin, comprising the steps of: (a) providing a first solution in which a taxane is dissolved at 55-75% (w / v); (b) preparing a second solution in which albumin is dissolved at 15-25% (w / v); (c) mixing the first solution and the second solution to form a suspension; and (d) diluting the suspension with an aqueous solvent in an amount of 3 to 12 times the volume of the suspension.

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