Nanoparticles comprising sirolimus and albumin, pharmaceutical composition for subcutaneous administration, comprising same, and preparation method therefor

Abstract

The present invention relates to nanoparticles comprising sirolimus and albumin, a pharmaceutical composition for subcutaneous administration, comprising same, and a preparation method therefor. If the preparation method of the present invention is used, nanoparticles comprising sirolimus and albumin and having excellent improved particle size distribution maintenance stability can be prepared, and the prepared nanoparticles can be prepared in a pharmaceutical composition suitable for subcutaneous injection, and thus dosage is increased and convenience of administration is improved.

Description

Nanoparticles containing sirolimus and albumin, pharmaceutical composition for subcutaneous administration containing the same, and method for preparing the same

[0001] This invention is the result of an application filed with support from the '2023 Global Startup Commercialization Support Project' of Gyeonggi Province and the Gyeonggi Province Economic and Science Promotion Agency.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0170005, filed with the Korean Intellectual Property Office on November 29, 2023, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to nanoparticles comprising sirolimus and albumin, a pharmaceutical composition for subcutaneous administration comprising the same, and a method for preparing the same.

[0004]

[0005] Sirolimus is a conserved serine / threonine kinase that plays a central role in intracellular signaling. Activation of the mTOR pathway is associated with cell proliferation and survival, while inhibition of mTOR signaling leads to inflammation and apoptosis. Because dysregulation of the mTOR signaling pathway has been implicated in human diseases, including cancer and autoimmune disorders, mTOR inhibitors such as sirolimus are widely used to treat solid tumors, hematologic malignancies, organ transplantation, restenosis, and rheumatoid arthritis. Sirolimus, also known as rapamycin, is marketed as Rapamune tablets and is used to prevent organ rejection in kidney transplant recipients and to treat lymphangioleiomyomatosis. Because it is a poorly soluble drug, its bioavailability (BA) is very low at 14%, and it is available in tablet and intravenous liquid formulations. However, in the case of intravenous injection formulations, the time required for administration is from 30 minutes to 60 to 24 hours, so there is a problem of low patient compliance, and there is a disadvantage of high initial blood concentration, which increases the intensity of side effects.

[0006]

[0007] The present inventors have diligently researched and developed a nanoparticle formulation of sirolimus that addresses the aforementioned issues. As a result, they discovered that combining sirolimus with albumin and adding hyaluronidase to produce nanoparticles yields high yields, exhibits minimal size change upon reconstitution in aqueous solvents, and is suitable for subcutaneous injection. This discovery led to the completion of the present invention.

[0008] Accordingly, an object of the present invention is to provide a method for producing nanoparticles comprising sirolimus and albumin.

[0009] Another object of the present invention is to provide nanoparticles comprising sirolimus, albumin, and hyaluronidase.

[0010] Another object of the present invention is to provide a pharmaceutical composition for treating cancer, comprising the nanoparticles described above.

[0011]

[0012] The present invention provides the following inventions 1 to 22.

[0013] 1. A method for manufacturing nanoparticles comprising sirolimus and albumin, comprising the following steps:

[0014] (a) a step of preparing a first solution in which sirolimus is dissolved at 5 to 75 w / v%;

[0015] (b) a step of preparing a second solution in which albumin is dissolved at 5 to 30 w / v%;

[0016] (c) a step of mixing and stirring the first solution and the second solution in a volume ratio of 1:5 to 1:70; and

[0017] (d) A step of diluting the solution obtained in step (c) with an aqueous solvent of 2 to 10 times its volume.

[0018] 2. A method for producing nanoparticles, further comprising the step of (e) drying the diluted solution under reduced pressure to remove the organic solvent.

[0019] 3. A method for producing nanoparticles, wherein in 2, the reduced pressure drying is performed under temperature conditions in the range of 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.

[0020] 4. A method for producing nanoparticles, wherein in 2 or 3, the reduced pressure drying is performed under pressure conditions in the range of 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.

[0021] 5. A method for producing nanoparticles, further comprising a step of filtering and drying the solution obtained through step (e) in any one of steps 1 to 4 to obtain nanoparticles.

[0022] 6. A method for producing nanoparticles, wherein in any one of 1 to 5, the drying is freeze-drying, and a freeze-drying protection agent is added to the freeze-drying process.

[0023] 7. A method for manufacturing nanoparticles, wherein hyaluronidase is added to the freeze-drying process in 6.

[0024] 8. A method for producing nanoparticles, wherein in any one of 1 to 7, the albumin is selected from the group consisting of human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), recombinant albumin (rHSA), and combinations thereof.

[0025] 9. A method for producing nanoparticles, wherein in any one of 1 to 8, the step of mixing and stirring the first solution and the second solution (c) includes a high-pressure homogenization process.

[0026] 10. A method for producing nanoparticles, wherein the high-pressure homogenization in 9 is performed under pressure conditions of 5,000 to 30,000 psi.

[0027] 11. A method for producing nanoparticles, wherein in any one of 1 to 10, the solvent of the first solution is selected from the group consisting of 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.

[0028] 12. A method for producing nanoparticles, wherein in any one of 1 to 11, the solvent of the second solution is an aqueous solvent selected from the group consisting of water, distilled water, sterilized 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.

[0029] 13. A method for producing nanoparticles, wherein step (d) is performed at a temperature of 1 to 40°C in any one of steps 1 to 12.

[0030] 14. Nanoparticles comprising sirolimus, albumin, and hyaluronidase.

[0031] 15. In 14, the nanoparticles are electrostatically bound to each other by sirolimus and albumin.

[0032] 16. Nanoparticles according to 14 or 15, wherein the weight ratio of sirolimus and albumin is 1:5 to 1:20.

[0033] 17. A nanoparticle according to any one of 14 to 16, further comprising a freeze-drying protection agent.

[0034] 18. A nanoparticle according to any one of 14 to 17, wherein the nanoparticle is manufactured by the manufacturing method of 1 to 13.

[0035] 19. A nanoparticle according to any one of 14 to 18, wherein the albumin is selected from the group consisting of human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), recombinant human serum albumin (rHSA), and combinations thereof.

[0036] 20. A pharmaceutical composition for treating cancer, comprising any one of the nanoparticles of 14 to 19.

[0037] 21. In 20, the pharmaceutical composition is a subcutaneous administration formulation, and is a pharmaceutical composition for the purpose of treating cancer or as an immunosuppressant.

[0038] 22. A method for treating cancer, comprising administering to a subject in need of treatment a nanoparticle of any one of 14 to 19; or a pharmaceutical composition of 20 or 21.

[0039]

[0040] According to one aspect of the present invention, the present invention provides a method for preparing nanoparticles comprising sirolimus and albumin, comprising the following steps:

[0041] (a) a step of preparing a first solution in which sirolimus is dissolved at 5 to 75 w / v%;

[0042] (b) a step of preparing a second solution in which albumin is dissolved at 5 to 30 w / v%;

[0043] (c) a step of mixing and stirring the first solution and the second solution in a volume ratio of 1:5 to 1:70; and

[0044] (d) A step of diluting the solution obtained in step (c) with an aqueous solvent of 2 to 10 times its volume.

[0045]

[0046] The term "about" used in this specification in relation to numerical limitations, such as temperature, time, pressure, amount, concentration, etc., including ranges, can mean a range or specific number of (+) or (-) 10%, 5%, 4%, 3%, 2%, 1%, or any number therebetween.

[0047] As used herein, the term "nanoparticle" refers to a particle having a particle size of less than 1 μm. When the nanoparticle of the present invention is intended for intravenous injection, the nanoparticle preferably has a size of 500 nm or less. Specifically, the nanoparticles have a size of 50-500 nm, 50-400 nm, 50-300 nm, 50-200 nm, 50-190 nm, 50-180 nm, 50-170 nm, 50-160 nm, 50-150 nm, 80-500 nm, 80-400 nm, 80-300 nm, 80-200 nm, 80-190 nm, 80-180 nm, 80-170 nm, 80-160 nm, 80-150 nm, 100-500 nm, 100-400 nm, 100-300 nm, 100-200 nm, 100-190 nm, 100-180 nm, 100-170 nm, It is preferable to have a size of 100-160 nm, 100-150 nm, 110-500 nm, 110-400 nm, 110-300 nm, 110-200 nm, 110-190 nm, 110-180 nm, 110-170 nm, 110-160 nm, or 110-150 nm.

[0048] In one embodiment of the present invention, the concentration of sirolimus in the first solution is, for example, 5 to 75% (w / v), 5 to 70%, 5 to 65%, 5 to 60% (w / v), 5 to 55% (w / v), 5 to 50% (w / v), 5 to 45% (w / v), 5 to 40% (w / v), 5 to 35% (w / v), 5 to 30% (w / v), 5 to 25% (w / v), 5 to 20% (w / v), 5 to 15% (w / v), 5 to 10% (w / v), 10 to 75% (w / v), 15 to 75% (w / v), 20 to 75% (w / v), 25 to 75% (w / v), 30 to 75 %(w / v), 35 to 75 %(w / v), 40 to 75 %(w / v), 45 to 75 %(w / v), 50 to 75 %(w / v), 55 to 75 %(w / v), 60 to 75 %(w / v), 65 to 75 %(w / v), 70 to 75 %(w / v), 10 to 65 %(w / v), 15 to 60 %(w / v), 20 to 55 %(w / v), or 25 to 50 %(w / v), 30 to 45 %(w / v), but is not limited thereto. Preferably, it may be 10 to 60%.

[0049] In one embodiment of the present invention, the solvent of the first solution is selected from the group consisting of 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, but is not limited thereto.

[0050] In one embodiment of the present invention, when the concentration of sirolimus in the first solution is less than the lower limit of the range of 5 to 75% (w / v) defined in the present invention, a problem occurs in which the average particle size upon rehydration rapidly increases to 200 nm or more even when the water dilution step is performed.

[0051] In addition, if the concentration of sirolimus in the first solution exceeds the upper limit of the range of 5 to 75% (w / v) defined in the present invention, sirolimus precipitates during the nanoparticle manufacturing process, making normal operation of the high-pressure homogenizer impossible, and the particle size of the nanoparticles also increases, causing a problem in that nanoparticles with a uniform particle size cannot be produced when the concentration exceeds 75% (w / v).

[0052] In one embodiment of the present invention, the concentration of albumin in the second solution may be, for example, 5 to 30% (w / v), 5 to 25% (w / v), 5 to 20% (w / v), 5 to 15% (w / v), 5 to 10% (w / v), 10 to 30% (w / v), 15 to 30% (w / v), 20 to 30% (w / v), 25 to 30% (w / v), 10 to 25% (w / v), 15 to 20% (w / v), but is not limited thereto.

[0053] 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, sterilized 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 thereto.

[0054] When the albumin concentration of the second solution is less than the lower limit of the range of 5 to 30% (w / v) defined in the present invention, the volume of the dispersion in the final nanoparticles is excessively increased, and accordingly, the filling amount becomes so large that it is impossible to produce commercially due to the low content. As a result, since the normal filling volume range is exceeded, an additional concentration process is required, which causes problems of reduced economy and efficiency. In addition, when the albumin concentration of the second solution is more than the upper limit of the range of 5 to 30% (w / v) defined in the present invention, even if a water dilution step is performed, a problem occurs in which the average particle size upon rehydration rapidly increases to more than 200 nm.

[0055] The volume ratio of the first solution containing the above-mentioned sirolimus dissolved and the second solution containing the above-mentioned albumin dissolved may be 1:5 to 1:70. For example, the volume ratio of the first solution and the second solution is 1: 5 to 1: 70, 1: 5 to 1: 65, 1: 5 to 1: 60, 1: 5 to 1: 55, 1: 5 to 1: 50, 1: 5 to 1: 45, 1: 5 to 1: 40, 1: 5 to 1: 35, 1: 5 to 1: 30, 1: 5 to 1: 25, 1: 5 to 1: 20, 1: 5 to 1: 15, 1: 5 to 1: 10, 1: 10 to 1: 70, 1: 15 to 1: 70, 1: 20 to 1: 70, 1: 25 to 1: 70, 1: It may be, but is not limited to, 30 to 1:70, 1:35 to 1:70, 1:40 to 1:70, 1:45 to 1:70, 1:50 to 1:70, 1:55 to 1:70, 1:60 to 1:70, 1:65 to 1:70, 1:10 to 1:65, 1:15 to 1:60, 1:20 to 1:55, 1:25 to 1:50, or 1:30 to 1:45.

[0056] Considering the reactivity and stability of the nanoparticles finally manufactured, the volume ratio of the first solution and the second solution can be appropriately selected so that the weight ratio of sirolimus:albumin is 1:5 to 1:20. For example, the weight ratio of sirolimus:albumin is 1:5 to 1:20, 1:5 to 1:18, 1:5 to 1:16, 1:5 to 1:14, 1:5 to 1:12, 1:5 to 1:10, 1:5 to 1:8, 1:7 to 1:20, 1:9 to 1:20, 1:11 to 1:20, 1:13 to 1:20, 1:15 to 1:20, 1:17 to 1:20, 1:6 to 1:17, 1:6.5 to 1:14, 1:7 to 1:11, 1:5 to 1:20, 1:5 to 1:20, 1:5 to 1:20, It may be, but is not limited to, 1:5 to 1:20, 1:5 to 1:20, 1:5 to 1:20, 1:5 to 1:20, 1:5 to 1:20, 1:5 to 1:20.

[0057] In one embodiment of the present invention, the dilution in step (d) may be diluted 2 to 12 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, 2 to 4 times, 4 to 12 times, 6 to 12 times, 8 to 12 times, 10 to 12 times, 4 to 10 times, or 6 to 8 times based on the volume of the suspension, but is not limited thereto. Preferably, it may be 2 to 10 times.

[0058] If the amount of aqueous solvent used for dilution is less than the lower limit of the 2 to 10 times range defined in the present invention, a problem occurs in which the particle size rapidly increases to 200 nm or more before drying under reduced pressure even after performing the water dilution step. This increase in particle size may be due to agglomeration between nanoparticles. If nanoparticles aggregate, it may affect the efficacy and safety of a pharmaceutical composition containing nanoparticles. Accordingly, a process for removing aggregates is required, which immediately leads to a decrease in yield, thereby reducing economic feasibility and efficiency.

[0059] In addition, when the amount of water-based solvent used exceeds the upper limit of the range of 2 to 10 times defined in the present invention, not only does the unencapsulated rate of sirolimus increase, but more water is used than necessary to stabilize the nanoparticle size, which causes problems in efficiency and economy in terms of the scale-up process.

[0060] In one embodiment of the present invention, the step (d) may be performed at a temperature condition of 1 to 40°C. For example, the temperature may be 1 to 40°C, 1 to 35°C, 1 to 30°C, 1 to 25°C, 1 to 20°C, 1 to 15°C, 1 to 10°C, 1 to 5°C, 5 to 40°C, 10 to 40°C, 15 to 40°C, 20 to 40°C, 25 to 40°C, 30 to 40°C, 35 to 40°C, 5 to 35°C, 10 to 30°C, or 15 to 25°C, but is not limited thereto.

[0061] In one embodiment of the present invention, the manufacturing method further includes the step of (e) drying the diluted solution under reduced pressure to remove the organic solvent.

[0062] In the case where the drying according to one embodiment of the present invention is reduced pressure drying, it can be performed by adopting a conventional reduced pressure drying method, such as using a rotary evaporator, and is not limited to specific reduced pressure drying conditions.

[0063] In a specific embodiment of the present invention, the reduced pressure drying may be performed under temperature conditions ranging from 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, but is not limited thereto.

[0064] In a specific embodiment of the present invention, the reduced pressure drying may be performed under pressure conditions in the range of 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, but is not limited thereto.

[0065] The nanoparticles present in the above diluted solution can maintain an average particle size of 100 to 200 nm, preferably 110 to 190 nm, from before drying to the completion of drying.

[0066] In one specific example of the present invention, the nanoparticles present in the diluted solution maintain an average particle size of 60 to 200 nm from before drying is performed until drying is completed.

[0067] In one specific example of the present invention, the nanoparticles present in the diluted solution maintain an average particle size of 70 to 190 nm from before drying to the completion of drying.

[0068] In one embodiment of the present invention, the sirolimus has a binding degree of 80% or more with albumin. The binding degree between sirolimus and albumin can be measured by a person skilled in the art using a technique such as chromatography, centrifugation, or surface plasmon resonance to measure binding degree.

[0069] In one embodiment of the present invention, the albumin is selected from the group consisting of human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), recombinant albumin (rHSA), and combinations thereof.

[0070] In one embodiment of the present invention, the step of mixing and stirring the first solution and the second solution (c) includes a high-pressure homogenization process.

[0071] In one specific example of the present invention, the high-pressure homogenization is performed under pressure conditions of 5,000 to 30,000 psi.

[0072] In one specific embodiment of the present invention, the high pressure homogenization is performed 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, 16,000 to 25,000 psi, 17,000 to 25,000 psi, 18,000 to 25,000 psi, 10,000 to It is performed at conditions of, but not limited 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.

[0073] The above high pressure homogenization is performed one or more times, more specifically one to five times, one to four times, or one to three times, but is not limited thereto.

[0074] In one embodiment of the present invention, the manufacturing method further includes a step of filtering and drying the solution obtained through step (e) to obtain nanoparticles.

[0075] In a specific embodiment of the present invention, the drying is freeze-drying.

[0076] In one embodiment of the present invention, the freeze-drying process involves freezing nanoparticles, the target material for drying, and then removing the frozen solvent by sublimation in a vacuum environment. The freeze-drying process may optionally include excipients or freeze-drying protectants to enhance the storage stability of the freeze-dried product.

[0077] The excipients or lyoprotectants include, but are not limited to, polymers such as dextran and polyethylene glycol; sugars such as mannitol, sucrose, glucose, trehalose, and lactose; surfactants such as polysorbates; and amino acids such as glycine, arginine, and serine, preferably mannitol, sucrose, or trehalose.

[0078] In one embodiment of the present invention, hyaluronidase is added to the freeze-drying process.

[0079] In the present invention, the term 'hyaluronidase' refers to an enzyme that can break down hyaluronic acid, a main component of the extracellular matrix, and although its half-life is very short at 2 minutes, its duration of effect is long at 24-48 hours, so it increases the drug administration dose when injected subcutaneously and helps absorption. The hyaluronidase is derived from mammals, bacteria, leeches, and other parasites. One type of hyaluronidase is pH20 derived from mammals, and in the case of intact human-derived pH20, it has the characteristic of being fixed to the cell membrane due to a GPI (glycosylphosphatidylinositol) anchor and is insoluble. Therefore, mammalian-derived hyaluronidase is mainly sold as a liquid or lyophilized extract of sheep or cow testis and is manufactured at 1,500 U / ml. Since it is of animal origin, it may contain nonhuman proteins, etc., which may cause an immune response. Therefore, recombinant human hyaluronidase may be used. The recombinant human hyaluronidase is a hyaluronidase that has been improved with human recombinant genes, and is produced in animal cells such as genetically engineered Chinese Hamster Ovary (CHO) cells. It is soluble and has activity at neutral and acidic pH, so it has wide versatility. In addition, compared to animal-derived hyaluronidase, it can be produced at a high activity concentration of 100,000 U / mg or more.

[0080]

[0081] According to another aspect of the present invention, the present invention provides nanoparticles comprising sirolimus, albumin, and hyaluronidase.

[0082] The nanoparticle according to one aspect of the present invention can be manufactured by the above-described method for manufacturing nanoparticles.

[0083] In one embodiment of the present invention, sirolimus and albumin are combined with each other.

[0084] In one embodiment of the present invention, the weight ratio of sirolimus and albumin is 1:5 to 1:20, but is not limited thereto.

[0085] In one embodiment of the present invention, the nanoparticles additionally comprise a lyophilization protectant. The lyophilization protectant is as described above.

[0086]

[0087] In one embodiment of the present invention, the nanoparticles may be pharmaceutically formulated for administration to a patient. Accordingly, in another aspect of the present invention, a pharmaceutical composition for treating cancer comprising the nanoparticles is provided.

[0088] The above pharmaceutical composition can be used for the treatment of cancers in which the active ingredient sirolimus is used. The cancers include, but are not limited to, perivascular epithelioid cell tumor (PEComa).

[0089] Various formulations and drug delivery systems are available in the art for the formulation of the above pharmaceutical compositions. For example, see Gennaro, AR, ed. (1995) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.

[0090] In one embodiment of the present invention, the nanoparticles of the present invention can be administered as a pharmaceutical composition by one of the following routes: orally; parenterally, such as transdermally, intranasally, intramuscularly, intravenously, subcutaneously, intradermally, intratumorally, etc.

[0091] In one embodiment of the present invention, the nanoparticles of the present invention may include the following solid, liquid, semi-solid, or gaseous excipients.

[0092] The above 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.

[0093] The liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and various oils including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly those for injectable solutions, include water, saline, aqueous dextrose, and glycols. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, edited by E.W. Martin (Mack Publishing Company, 18th ed., 1990).

[0094] In a specific embodiment of the present invention, the nanoparticles may be formulated for subcutaneous injection.

[0095]

[0096] The features and advantages of the present invention are summarized as follows:

[0097] (a) The present invention provides a method for preparing nanoparticles comprising sirolimus and albumin. (b) The present invention provides nanoparticles comprising sirolimus, albumin, and hyaluronidase. (c) The present invention provides a pharmaceutical composition for treating cancer comprising the nanoparticles.

[0098] When the manufacturing method of the present invention is used, nanoparticles containing sirolimus and albumin with excellent particle size distribution maintenance stability can be manufactured, and the manufactured nanoparticles can be manufactured into a pharmaceutical composition suitable for subcutaneous injection, thereby improving the administration dosage and convenience of administration.

[0099]

[0100] Figure 1 is a diagram showing the concentration of sirolimus in the blood after intravenous or subcutaneous injection to confirm the pharmacokinetic information of sirolimus and albumin nanoparticles of the present invention.

[0101]

[0102] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0103]

[0104] Example

[0105]

[0106] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0107]

[0108] Example 1: Preparation of nanoparticles containing sirolimus and albumin

[0109] Nanoparticles containing sirolimus and albumin were prepared as follows.

[0110] More specifically, 3.17 g of sirolimus was dissolved in chloroform to prepare a sirolimus solution (first solution) with a concentration of 30% w / v. In addition, a 12% albumin solution (second solution, Greencross Human Serum Albumin Inj. 20%) was prepared by diluting an albumin solution with a concentration of 20% w / v. The first solution was added to the prepared second solution, and the mixture was mixed in a high-speed homogenizer (IKA, Germany) at 5,000 to 10,000 rpm for 5 minutes. The mixture was transferred to a high-pressure homogenizer and high-pressure homogenized for 2 to 3 cycles at 5,000 to 15,000 psi. The high-pressure homogenized mixture was diluted with 6 to 7 times the volume of water.

[0111] Nanoparticles were manufactured by performing the same method as described above, but varying the concentration of the first or second solution and the amount of water used for dilution. The specific variable conditions for each example are shown in Table 1 below.

[0112]

[0113] Method for manufacturing nanoparticles containing sirolimus and albumin according to conditions Example 1 Solution concentration, w / v % Second solution concentration, w / v % Water usage ratio 1-110.012.061-220.01-330.01-440.01-550.01-660.02-1305.062-210.02-315.02-420.02-530.03-13012.04.03-26.03-38.0

[0114] Example 2. Particle size trends of nanoparticles by process

[0115] A sirolimus solution (the first solution) with a 44% w / v concentration was prepared by dissolving 4.26 g of sirolimus in chloroform. In addition, an albumin solution with a 20% w / v concentration was diluted to prepare an albumin solution (the second solution, Greencross Human Serum Albumin Inj. 20%). The first solution was added to the prepared second solution, and the mixture was mixed in a high-speed homogenizer (IKA, Germany) at 5,000 to 10,000 rpm for 5 minutes. Subsequently, the mixture was transferred to a high-pressure homogenizer and subjected to high-pressure homogenization for 2 to 3 cycles at 5,000 to 15,000 psi. The high-pressure homogenized mixture was diluted with 6 to 7 times the volume of water.

[0116] After dilution, the organic solvent was removed by drying under reduced pressure using a rotary evaporator under pressure conditions ranging from 20 to 50°C and 30 to 60 hPa.

[0117] The dispersion liquid, from which the organic solvent was removed through decompression drying, was filtered through a sterile filtration (0.22 um filter) and then filled into a vial for freeze-drying.

[0118] To determine whether particle size was formed and the stability of each process, particle sizes were measured for the diluted solution of the high-pressure homogenized mixture, the solution dried under reduced pressure, the solution aseptically filtered, and the solution rehydrated after freeze-drying. Particle size was measured using a Zetasizer from Marlvern, using the light scattering method (laser angle 90 degrees, temperature 20°C, and solvent distilled water).

[0119]

[0120] Particle size results by manufacturing process of sirolimus and albumin nanoparticles Process Z-average (nm) PDIPeak 1 (volume) High-pressure homogenized mixture diluted with water 151.7 0.140 143.5 Liquid dried under reduced pressure 154.9 0.132 147.7 Liquid sterile filtered 146.6 0.117 135.6 Rehydrated liquid after freeze-drying 191.10 136 193.0

[0121] As a result of measuring particle size, it was confirmed that the particle size increased significantly when freeze-drying was performed after sterile filtration.

[0122] Afterwards, several freeze-dried samples were rehydrated and the particle size was measured as shown in Table 3 below to check the particle size trend and deviation between samples.

[0123]

[0124] Particle size results after freeze-drying of sirolimus and albumin nanoparticles No. Z-average (nm) PDIPeak 1 (volume) 1172.00.096 171.92 186.50.126 193.13 177.80.127 179.04 187.20.119 190.85 186.90.129 191.26 186.30.083 190.87 170.10.110 168.18 176.30.123 179.9

[0125] The particle size of nanoparticles in the rehydrated solution after freeze-drying was confirmed to increase by approximately 30 to 50 nm compared to the sterile-filtered solution before freeze-drying. Furthermore, variations were observed between simultaneously freeze-dried samples.

[0126]

[0127] Example 3. Preparation of sirolimus and albumin nanoparticles mixed with a lyophilization protectant

[0128] A sirolimus solution (solution 1) and an albumin solution (solution 2) dissolved in chloroform were mixed in a high-speed homogenizer (IKA, Germany) at 5,000–10,000 rpm for 5 minutes. The mixture was then transferred to a high-pressure homogenizer and subjected to two cycles of high-pressure homogenization at 5,000–15,000 psi. The mixture after high-pressure homogenization was diluted with water.

[0129] After dilution, the organic solvent was removed by drying under reduced pressure using a rotary evaporator.

[0130] The dispersion from which the organic solvent was removed through vacuum drying was filtered aseptically, and then freeze-drying was performed by dissolving mannitol, sucrose, and trehalose, which are freeze-drying protectants, in a concentration of 2 to 10% w / v and filling the vial into a glass vial.

[0131] The particle size of the liquid that underwent sterile filtration was measured, and the particle size of the freeze-dried sample was measured according to the presence or absence of a freeze-drying protection agent.

[0132] Particle size results of lyophilized samples with and without freeze-drying protectant Item Z-average (nm) PDI Preak 1 (volume) Aseptic filtered solution 166.9 0.125 165.5 Without freeze-drying protectant 191.00.125 201.7 Mannitol 2% 167.7 0.125 162.5 Mannitol 5% 165.2 0.148 159.9 Mannitol 10% 166.2 0.112 162.5 Sucrose 2% 169.7 0.134 165.3 Sucrose 5% 167.5 0.122 162.6 Sucrose 10% 167.3 0.125 161.6 Trehalose 2% 171.00.115 170.0 Trehalose 5%167.00.101164.3 Trehalose 10%168.10.102165.9

[0133] The measurement results confirmed that there was no tendency for particle size to increase when freeze-drying protectants were mixed, except for the 2% concentration of sucrose and trehalose.

[0134]

[0135] Example 4. Preparation of sirolimus and albumin nanoparticles mixed with a lyophilized protectant and hyaluronidase.

[0136] Nanoparticles containing sirolimus, albumin, and hyaluronidase were prepared as follows.

[0137] More specifically, 3.17 g of sirolimus was dissolved in 4.3% chloroform to prepare a 30% w / v sirolimus solution (first solution). In addition, a 20% w / v albumin solution was diluted to prepare a 12% albumin solution (second solution, Greencross Human Serum Albumin Inj.). The first solution was added to the prepared second solution, and the mixture was mixed in a high-speed homogenizer (IKA, Germany) at 5,000 to 10,000 rpm for 5 minutes. Subsequently, the mixture was transferred to a high-pressure homogenizer and high-pressure homogenized for 2 to 3 cycles at 5,000 to 15,000 psi. The high-pressure homogenized mixture was diluted with 6 to 7 times the volume of water.

[0138] After dilution, drying under reduced pressure is performed using a rotary evaporator, and the organic solvent is removed under pressure conditions ranging from 20 to 50°C and 30 to 60 hPa.

[0139] The dispersion from which the organic solvent has been removed through vacuum drying is aseptically filtered, dissolved with a lyophilization protectant (mannitol, sucrose, trehalose) at a concentration of 3 to 10% w / v, and filled into a vial. Animal-derived hyaluronidase is added to the vial at a concentration of 2000 U / ml (10,000 U per vial), 4000 U / ml (20,000 U per vial), etc., and lyophilization is performed.

[0140]

[0141] Particle size results of lyophilized samples with and without lyophilization protectant and hyaluronidase. Item Z-average (nm) PDIPeak 1 (volume) Mannitol without hyaluronidase 117.3 0.15 5 9 6.3 Mannitol 5%, Hyaluronidase without 107.3 0.10 4 9 4.6 Mannitol 5%, Hyaluronidase 2000 U / ml 131.7 0.15 7 115.0 Mannitol 5%, Hyaluronidase 4000 U / ml 110.8 0.13 2 9 5.1

[0142] As shown above, it was confirmed that the particle size increased rapidly after lyophilization when the lyophilization protectant mannitol was not used. In addition, it was confirmed that the particle size after lyophilization remained smaller when the concentration of hyaluronidase was 4000 U / ml, which is higher than 2000 U / ml.

[0143]

[0144] Example 5. Pharmacokinetic study of sirolimus and albumin nanoparticles mixed with lyophilized protectant and hyaluronidase.

[0145] The present inventors manufactured albumin nanoparticles containing a lyophilized protective agent (mannitol 5%) and hyaluronidase (0, 2000, 4000 U / ml) using the same method as in Example 4, and performed pharmacokinetic tests according to the route of administration as follows.

[0146] PK experimental information of sirolimus and albumin nanoparticles of the present invention Group (n) Drug concentration Drug dose (Sirolimus, mg / kg) Drug dosage (mL / kg) Administration route Sirolimus (mg / mL) Hyaluronidase (U / mL) G1 (3) 2021 Intravenous injection (IV) G2 (3) Subcutaneous injection (SC) G3 (3) 2,000 G4 (3) 4,000

[0147] For group 1, SD rats were injected intravenously, while groups 2 to 4 were injected subcutaneously. Blood samples were collected at each time point to measure the concentration of sirolimus in the whole blood. The results are shown in Table 7 and Fig. 1.

[0148]

[0149] Pharmacokinetic parameters by group of sirolimus and albumin nanoparticles of the present invention (mean value ± standard deviation) Pharmacokinetic parameters G1 (IV) G2 (SC) (hyaluronidase not administered) G3 (SC) G4 (SC) Hyaluronidase low dose Hyaluronidase high dose C0 (ng / mL) 235.7 ± 9.2 NANANAC max (ng / mL)NA58.9 ± 9.5110.9 ± 17.7119.4 ± 32.0T max (h)0.3 ± 0.02.7 ± 1.21.2 ± 0.81.7 ± 0.6t 1 / 2 (h)14.0 ± 1.09.4 ± 0.711.3 ± 1.110.2 ± 1.6AUC last (ngㆍh / mL)660.8 ± 84.7703.9 ± 83.0748.9 ± 118.8790.6 ± 52.7Relative AUC (%)100.0106.5113.3119.6Relative C max (ng / mL)NA100.0188.4202.9

[0150] As shown in Table 7 and Fig. 1, the drug exposure according to the difference in administration route at the same dosage was increased by 6.5% in Relative AUC for G2 (subcutaneous injection) compared to G1 (intravenous injection), and the drug exposure according to the amount of added hyaluronidase at the same dosage and administration route was increased by 6.8% in Relative AUC for G3 (with hyaluronidase) compared to G2 (without hyaluronidase) and by 13.1% in G4 (with high dose hyaluronidase) compared to G2. This showed a slightly different trend compared to the data for Herceptin hylecta. According to 'NDA / BLA Multi-Disciplinary Review and Evaluation (761106)', in the case of Herceptin hylecta, Tmax is delayed (24-29h vs 67h) compared to the formulation that does not use hyaluronidase, and Cmax or AUC are not significantly affected. However, what can be clearly confirmed in the examples of the present invention is that in the case of the nanoparticles of the present invention mixed with hyaluronidase, Tmax is delayed, Cmax and AUC increase, and the graph shows a graph pattern in which the initial burst type is slowly released compared to IV.

[0151] Therefore, based on the above results, it is believed that hyaluronidase can relatively increase bioavailability compared to non-hyaluronidase use, and that the Cmax is significantly lower than that of the existing IV route, thereby reducing adverse drug reactions. Considering these results comprehensively, hyaluronidase can be effectively used as a subcutaneous injection with high administration convenience and patient compliance.

Claims

1. A method for producing nanoparticles comprising sirolimus and albumin, comprising the following steps: (a) a step of preparing a first solution in which sirolimus is dissolved at 5 to 75 w / v%; (b) a step of preparing a second solution in which albumin is dissolved at 5 to 30 w / v%; (c) a step of mixing and stirring the first solution and the second solution having a volume ratio of 1:5 to 1:70; and (d) A step of diluting the solution obtained in step (c) with an aqueous solvent of 2 to 10 times its volume.

2. A method for producing nanoparticles, in accordance with claim 1, further comprising the step of (e) drying the diluted solution under reduced pressure to remove the organic solvent.

3. A method for producing nanoparticles, further comprising a step of filtering and drying the solution obtained through step (e) in paragraph 2 to obtain nanoparticles.

4. A method for manufacturing nanoparticles, wherein in the third paragraph, the drying is freeze-drying, and a freeze-drying protection agent is added to the freeze-drying process.

5. A method for manufacturing nanoparticles, wherein in the 4th paragraph, hyaluronidase is added to the freeze-drying process.

6. A method for producing nanoparticles in the first paragraph, wherein the albumin is selected from the group consisting of human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin (OVA), recombinant human serum albumin (rHSA), and combinations thereof.

7. A method for producing nanoparticles in claim 1, wherein the step of mixing and stirring the first solution and the second solution (c) includes a high-pressure homogenization process.

8. A method for producing nanoparticles, wherein in clause 7, the high-pressure homogenization is performed under pressure conditions of 5,000 to 30,000 psi.

9. Nanoparticles comprising sirolimus, albumin, and hyaluronidase.

10. A nanoparticle in claim 9, wherein sirolimus and albumin are electrostatically bound to each other.

11. A nanoparticle in claim 9, wherein the weight ratio of sirolimus and albumin is 1:5 to 1:

20.

12. A nanoparticle according to claim 9, further comprising a freeze-drying protection agent.

13. A pharmaceutical composition for treating cancer, comprising a nanoparticle according to any one of claims 9 to 12.

14. In claim 13, the pharmaceutical composition is a pharmaceutical composition for the purpose of treating cancer or as an immunosuppressant, which is a subcutaneous administration formulation.

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