Nanoparticles comprising sirolimus and albumin, pharmaceutical compositions for subcutaneous administration comprising the same and methods of preparation thereof

Abstract

The present invention relates to nanoparticles comprising sirolimus and albumin, a pharmaceutical composition comprising the same for subcutaneous administration, and a method for preparing the same. In the case of using the preparation method of the present invention, nanoparticles comprising sirolimus and albumin having excellent stability of particle size distribution can be prepared, the prepared nanoparticles can be prepared as a pharmaceutical composition suitable for subcutaneous injection, and the increase in administration dose and the convenience of administration can be improved.

Description

Technical Field

[0001] This invention was applied for with the support of the "2023 Global Startup Commercialization Support Project" of Gyeonggi Province and the Gyeonggi Economic Science Promotion Agency in South Korea.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0170005, filed on November 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to nanoparticles containing sirolimus and albumin, pharmaceutical compositions containing the same for subcutaneous administration, and methods for preparing the same. Background Technology

[0004] Sirolimus, also known as rapamycin, is the mammalian target of rapamycin (mTOR), 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. Abnormal regulation of the mTOR signaling pathway is associated with human diseases, including cancer and autoimmune disorders; therefore, mTOR inhibitors such as sirolimus are widely used in the treatment of solid tumors, hematologic malignancies, organ transplantation, restenosis, and rheumatoid arthritis. Sirolimus, also known as rapamycin, is marketed under the brand name rapamycin and is used for the prevention of organ rejection in kidney transplant patients and the treatment of lymphangioleiomyomas. Due to its poor solubility and low bioavailability (BA) of only 14%, it has been developed in tablet and intravenous liquid formulations. However, in the case of intravenous injection, the administration time can range from 30 minutes to 60 minutes or even 24 hours, which leads to low patient compliance and the disadvantage of high initial blood concentration causing severe side effects. Summary of the Invention

[0005] [Technical Issues]

[0006] To address the aforementioned problems, the inventors focused on developing a nanoparticle formulation of sirolimus. As a result, it was discovered that when sirolimus was combined with albumin and hyaluronidase was added to prepare nanoparticles, not only was the yield high, but the size of the nanoparticles did not change drastically during reconstruction in an aqueous solvent, remaining suitable for subcutaneous injection, thus completing this invention.

[0007] Therefore, the object of the present invention is to provide a method for preparing nanoparticles containing sirolimus and albumin.

[0008] Another object of the present invention is to provide nanoparticles comprising sirolimus, albumin and hyaluronidase.

[0009] Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the above-mentioned nanoparticles.

[0010] [Technical Solution]

[0011] The present invention provides the inventions described below 1 to 22.

[0012] 1. A method for preparing nanoparticles containing sirolimus and albumin, comprising the following steps:

[0013] Step (a): Prepare a first solution of sirolimus dissolved at a concentration of 5 w / v% to 75 w / v%.

[0014] Step (b): Prepare a second solution in which albumin is dissolved at a concentration of 5 w / v% to 30 w / v%.

[0015] Step (c) involves mixing and stirring the first and second solutions at a volume ratio of 1:5 to 1:70; and

[0016] Step (d) involves diluting the solution, based on the volume of the solution treated in step (c), with 2 to 10 times the volume of an aqueous solvent.

[0017] 2.1 The method for preparing nanoparticles containing sirolimus and albumin further includes step (e), which removes the organic solvent by drying the diluted solution under reduced pressure.

[0018] 3. In the preparation method of nanoparticles containing sirolimus and albumin as described in 2, the vacuum drying is carried out at temperatures ranging from 10°C to 70°C, 20°C to 70°C, 30°C to 70°C, 40°C to 70°C, 10°C to 60°C, 10°C to 50°C, 10°C to 40°C, 20°C to 60°C, 30°C to 50°C, 35°C to 45°C, 38°C to 42°C, or 39°C to 41°C.

[0019] 4. In the preparation method of nanoparticles containing sirolimus and albumin as described in 2 or 3, the reduced pressure drying is carried out under pressure conditions in the range of 10 hPa to 110 hPa, 20 hPa to 110 hPa, 30 hPa to 110 hPa, 40 hPa to 110 hPa, 50 hPa to 110 hPa, 10 hPa to 100 hPa, 10 hPa to 90 hPa, 10 hPa to 80 hPa, 10 hPa to 70 hPa, 20 hPa to 100 hPa, 30 hPa to 90 hPa, 40 hPa to 80 hPa, 50 hPa to 70 hPa, or 55 hPa to 65 hPa.

[0020] 5. The method for preparing nanoparticles containing sirolimus and albumin according to any one of 1 to 4 further includes step (f), filtering and drying the solution treated in step (e) to obtain nanoparticles.

[0021] 6. In any one of the methods for preparing nanoparticles containing sirolimus and albumin as described in 1 to 5, the drying is freeze-drying, and a freeze-drying protectant is added during the freeze-drying process.

[0022] 7. In the preparation method of nanoparticles containing sirolimus and albumin described in 6, hyaluronidase is added in the freeze-drying step.

[0023] 8. In any one of the methods for preparing nanoparticles containing sirolimus and albumin according to 1 to 7, 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.

[0024] 9. In any one of the methods for preparing nanoparticles containing sirolimus and albumin in any one of 1 to 8, step (c) of mixing and stirring the first solution and the second solution includes a high-pressure homogenization process.

[0025] 10. In the method for preparing nanoparticles containing sirolimus and albumin as described in 9, the high-pressure homogenization is carried out under pressure conditions of 5000 psi to 30000 psi.

[0026] 11. In any one of the methods for preparing nanoparticles containing sirolimus and albumin according to 1 to 10, the solvent of the first solution is selected from the group consisting of ethanol, methanol, isopropanol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone, and combinations thereof.

[0027] 12. In any one of the methods for preparing nanoparticles containing sirolimus and albumin according to 1 to 11, the solvent of the second solution is selected from the group consisting of water, distilled water, sterile water, phosphate buffered saline (PBS), methanol, pure water, ethanol, 1-propanol, 2-propanol, 1-pentanol, 2-butoxyethanol, ethylene glycol, acetone, 2-butanone, 4-methyl-2-pentanone, chloroform, and combinations thereof.

[0028] 13. In any one of the methods for preparing nanoparticles containing sirolimus and albumin, as described in 1 to 12, step (d) is carried out at a temperature of 1°C to 40°C.

[0029] 14. A nanoparticle comprising sirolimus, albumin and hyaluronidase.

[0030] 15. In the nanoparticles described in 14, the sirolimus and albumin are electrostatically bound together.

[0031] 16. In the nanoparticles described in 14 or 15, the weight ratio of sirolimus to albumin is 1:5 to 1:20.

[0032] 17. The nanoparticles in any one of 14 to 16 further contain a lyophilization protectant.

[0033] 18. In any one of 14 to 17, the nanoparticles are prepared by the preparation methods of 1 to 13.

[0034] 19. In any one of the nanoparticles from 14 to 18, the albumin is selected from the group consisting of human serum albumin, bovine serum albumin, ovalbumin, recombinant human serum albumin, and combinations thereof.

[0035] 20. A pharmaceutical composition for treating cancer, comprising any one of the nanoparticles described in 14 to 19.

[0036] 21. In the pharmaceutical composition of 20, the pharmaceutical composition is a subcutaneous dosage form for the treatment of cancer or as an immunosuppressant.

[0037] 22. A method of treating cancer, comprising the step of administering to a subject requiring treatment the nanoparticles described in any one of 14 to 19 or the pharmaceutical composition described in 20 or 21.

[0038] According to one embodiment of the present invention, the present invention provides a method for preparing nanoparticles containing sirolimus and albumin, comprising the following steps:

[0039] Step (a): Prepare a first solution of sirolimus dissolved at a concentration of 5 w / v% to 75 w / v%.

[0040] Step (b): Prepare a second solution in which albumin is dissolved at a concentration of 5 w / v% to 30 w / v%.

[0041] Step (c) involves mixing and stirring the first and second solutions at a volume ratio of 1:5 to 1:70; and

[0042] Step (d) involves diluting the solution, based on the volume of the solution treated in step (c), with 2 to 10 times the volume of an aqueous solvent.

[0043] The numerical limits used in this specification, such as the use of “about” in temperature, time, pressure, quantity, concentration, etc., including ranges, can indicate (+) or (-) 10%, 5%, 4%, 3%, 2%, 1%, or a range or specific value therebetween.

[0044] As used in this specification, the term "nanoparticle" refers to particles having a size of less than 1 μm. When the nanoparticles of the present invention are used for intravenous injection, preferably, the nanoparticles of the present invention have a size of less than 500 nm. Specifically, the nanoparticles have sizes 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, etc. Sizes of 00nm, 100-400nm, 100-300nm, 100-200nm, 100-190nm, 100-180nm, 100-170nm, 100-160nm, 100-150nm, 110-500nm, 110-400nm, 110-300nm, 110-200nm, 110-190nm, 110-180nm, 110-170nm, 110-160nm, or 110-150nm are preferred.

[0045] In one embodiment of the invention, for example, the concentration of sirolimus in the first solution may be 5% (w / v) to 75% (w / v), 5% (w / v) to 70% (w / v), 5% (w / v) to 65% (w / v), 5% (w / v) to 60% (w / v), 5% (w / v) to 55% (w / v), 5% (w / v) to 50% (w / v), 5% (w / v) to 45% (w / v), 5% (w / v) to 40% (w / v), 5% (w / v) to 35% (w / v), 5% (w / v) to 30% (w / v), 5% (w / v) to 25% (w / v), or 5% (w / v) to 20% (w / v). 5% (w / v) to 15% (w / v), 5% (w / v) to 10% (w / v), 10% (w / v) to 75% (w / v), 15% (w / v) to 75% (w / v), 20% (w / v) to 75% (w / v), 25% (w / v) to 75% (w / v), 30% (w / v) to 75% (w / v), 35% (w / v) to 75% (w / v), 40% (w / v) to 75% (w / v), 45% (w / v) to 75% (w / v), 50% (w / v) to 75% (w / v), 55% (w / v) to 75% (w / v), 60% (w / v) to 75% (w / v). The concentration can be 65% (w / v) to 75% (w / v), 70% (w / v) to 75% (w / v), 10% (w / v) to 65% (w / v), 15% (w / v) to 60% (w / v), 20% (w / v) to 55% (w / v), 25% (w / v) to 50% (w / v), or 30% (w / v) to 45% (w / v), but is not limited thereto. Preferably, it can be 10% to 60%.

[0046] In one embodiment of the present invention, the solvent of the first solution is selected from, but is not limited to, ethanol, methanol, isopropanol, butanol, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone, and combinations thereof.

[0047] In one embodiment of the present invention, if the concentration of sirolimus in the first solution is less than the lower limit of the range of 5% (w / v) to 75% (w / v) as defined in the present invention, then even if a water dilution step is performed, the average ion size will rapidly increase to more than 200 nm during rehydration.

[0048] Furthermore, if the concentration of sirolimus in the first solution is greater than the upper limit of the range of 5% (w / v) to 75% (w / v) defined in this invention, the high-pressure homogenizer will malfunction due to the precipitation of sirolimus during the nanoparticle preparation process. The particle size of the nanoparticles will also increase and exceed the concentration of 75% (w / v), resulting in the inability to produce nanoparticles with uniform particle size.

[0049] For example, in one embodiment of the present invention, the concentration of albumin in the second solution may be 5% (w / v) to 30% (w / v), 5% (w / v) to 25% (w / v), 5% (w / v) to 20% (w / v), 5% (w / v) to 15% (w / v), 5% (w / v) to 10% (w / v), 10% (w / v) to 30% (w / v), 15% (w / v) to 30% (w / v), 20% (w / v) to 30% (w / v), 25% (w / v) to 30% (w / v), 10% (w / v) to 25% (w / v), or 15% (w / v) to 20% (w / v), but is not limited thereto.

[0050] In one embodiment of the present invention, the solvent of the second solution is selected from the group consisting of water, distilled water, sterile water, phosphate buffered saline (PBS), methanol, pure water, ethanol, 1-propanol, 2-propanol, 1-pentanol, 2-butoxyethanol, ethylene glycol, acetone, 2-butanone, 4-methyl-2-pentanone, chloroform, and combinations thereof, but is not limited thereto.

[0051] If the albumin concentration in the second solution is less than the lower limit of the range of 50% (w / v) to 30% (w / v) defined in this invention, the volume of the dispersion in the final prepared nanoparticles will increase excessively. This low content will result in a filling volume exceeding what is acceptable for commercial production. Consequently, a concentration process will be required, exceeding the usual filling volume range, leading to reduced economic efficiency. Furthermore, if the albumin concentration in the second solution is greater than the upper limit of the range of 5% (w / v) to 30% (w / v) defined in this invention, even with a water dilution step, the average ion size will rapidly increase to over 200 nm during rehydration.

[0052] The volume ratio of the first solution for dissolving sirolimus to the second solution for dissolving albumin can be from 1:5 to 1:70. For example, the volume ratio of the first solution to the second solution can be 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, or 1:15 to 1:70. 1:20 to 1:70, 1:25 to 1:70, 1:30 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, but not limited to these.

[0053] Considering the reactivity and stability of the final prepared nanoparticles, the volume ratio of the first solution to the second solution can be appropriately selected so that the weight ratio of sirolimus to albumin is 1:5 to 1:20. For example, the weight ratio of sirolimus to albumin can be 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, or 1:1... 7 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, 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, but not limited to these.

[0054] In one embodiment of the invention, the dilution in step (d), based on the volume of the suspension, can be 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, but is not limited thereto. Preferably, it can be 2 to 10 times.

[0055] If the aqueous solvent used in the dilution is used in a quantity less than the lower limit of the range of 2 to 10 times defined in this invention, the average ion size will rapidly increase to over 200 nm before vacuum drying, even with the water dilution step. This increase in particle size occurs due to agglomeration between nanoparticles. Agglomeration of nanoparticles can affect the efficacy and safety of pharmaceutical compositions containing nanoparticles. Therefore, a process to remove agglomerates is required, which leads to a reduction in yield and consequently, reduced economic efficiency.

[0056] Furthermore, if the amount of aqueous solvent used exceeds the upper limit of the range of 2 to 10 times defined in this invention, it will not only increase the unencapsulated rate of sirolimus, but also require more water than is needed for nanoparticle size stabilization, thus causing inefficiency and economic problems in large-scale processes.

[0057] In one embodiment of the present invention, step (d) can be performed at a temperature of 1°C to 40°C. For example, the temperature conditions can 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°C to 40°C, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, 25°C to 40°C, 30°C to 40°C, 35°C to 40°C, 5°C to 35°C, 10°C to 30°C, or 15°C to 25°C, but are not limited thereto.

[0058] In one embodiment of the present invention, the preparation method further includes step (e), removing the organic solvent by drying the diluted solution under reduced pressure.

[0059] In the case of vacuum drying in one embodiment of the present invention, conventional vacuum drying methods such as rotary evaporators can be used, and the process is not limited to specific vacuum drying conditions.

[0060] In a specific embodiment of the present invention, the reduced pressure drying can be carried out at temperatures ranging from 10°C to 70°C, 20°C to 70°C, 30°C to 70°C, 40°C to 70°C, 10°C to 60°C, 10°C to 50°C, 10°C to 40°C, 20°C to 60°C, 30°C to 50°C, 35°C to 45°C, 38°C to 42°C, or 39°C to 41°C, but is not limited thereto.

[0061] In a specific embodiment of the present invention, the reduced pressure drying can be carried out under pressure conditions ranging from 10 hPa to 110 hPa, 20 hPa to 110 hPa, 30 hPa to 110 hPa, 40 hPa to 110 hPa, 50 hPa to 110 hPa, 10 hPa to 100 hPa, 10 hPa to 90 hPa, 10 hPa to 80 hPa, 10 hPa to 70 hPa, 20 hPa to 100 hPa, 30 hPa to 90 hPa, 40 hPa to 80 hPa, 50 hPa to 70 hPa, or 55 hPa to 65 hPa, but is not limited thereto.

[0062] The nanoparticles present in the diluted solution can maintain an average particle size of 100 nm to 200 nm from before drying to the end of drying, preferably 110 nm to 190 nm.

[0063] In one specific embodiment of the present invention, the nanoparticles present in the diluted solution maintain an average particle size of 60 nm to 200 nm from before the drying process to the end of the drying process.

[0064] In one specific embodiment of the present invention, the nanoparticles present in the diluted solution maintain an average particle size of 70 nm to 190 nm from before the drying process to the end of the drying process.

[0065] In one embodiment of the present invention, the binding degree of sirolimus to albumin is above 80%. The binding degree of sirolimus to albumin can be measured by methods such as chromatography, centrifugation, and surface plasmon resonance technology, which are commonly used by those skilled in the art to measure binding degree.

[0066] In one embodiment of the present invention, the albumin is selected from the group consisting of human serum albumin, bovine serum albumin, ovalbumin, recombinant human serum albumin, and combinations thereof.

[0067] In one embodiment of the present invention, step (c) of mixing and stirring the first solution and the second solution includes a high-pressure homogenization process.

[0068] In one specific embodiment of the invention, the high-pressure homogenization is performed under pressure conditions ranging from 5,000 psi to 30,000 psi.

[0069] In one specific embodiment of the invention, the high-pressure homogenization is performed at 10,000 psi to 30,000 psi, 12,000 psi to 30,000 psi, 15,000 psi to 30,000 psi, 16,000 psi to 30,000 psi, 17,000 psi to 30,000 psi, 18,000 psi to 30,000 psi, 10,000 psi to 25,000 psi, 12,000 psi to 25,000 psi, and 15,000 psi to 25,000 psi. The test may be conducted under conditions of 16,000 psi to 25,000 psi, 17,000 psi to 25,000 psi, 18,000 psi to 25,000 psi, 10,000 psi to 20,000 psi, 12,000 psi to 20,000 psi, 15,000 psi to 20,000 psi, 16,000 psi to 20,000 psi, 17,000 psi to 20,000 psi, or 18,000 psi to 20,000 psi, but is not limited to these conditions.

[0070] The high-pressure homogenization is performed more than once, more specifically, it can be performed 1 to 5 times, 1 to 4 times, or 1 to 3 times, but is not limited to this.

[0071] In one embodiment of the present invention, the preparation method further includes step (f), filtering and drying the solution treated in step (e) to obtain nanoparticles.

[0072] In a specific embodiment of the present invention, the drying is freeze-drying.

[0073] In one embodiment of the present invention, the freeze-drying refers to the process of freezing nanoparticles, the substance to be dried, and then removing the frozen solvent by sublimation in a vacuum environment. During the freeze-drying process, excipients or freeze-drying protectants may be selectively included to enhance the storage stability of the freeze-dried product.

[0074] The excipients or lyophilization protectants include: polymers, such as dextran and polyethylene glycol; sugars, such as mannitol, sucrose, glucose, trehalose, and lactose; surfactants, such as polysorbate; and amino acids, such as glycine, arginine, and serine. Mannitol, sucrose, or trehalose are preferred, but not limited thereto.

[0075] In one embodiment of the present invention, hyaluronidase is added during the freeze-drying process.

[0076] In this invention, the term "hyaluronidase" refers to an enzyme capable of breaking down hyaluronic acid, a major component of the extracellular matrix. It has an extremely short half-life of only 2 minutes, but its effects last for 24-48 hours (h). Therefore, increasing the dosage during subcutaneous injection aids absorption. The hyaluronidase is derived from mammals, bacteria, leeches, and other parasites. One example of such hyaluronidase is pH20 from atomic mammals. Complete human pH20 is immobilized in the cell membrane due to the GPI (glycosylphosphatidylinositol) linker and is insoluble. Therefore, mammalian-derived hyaluronidase is primarily an extract from sheep or bovine testes, sold in liquid or lyophilized form, prepared at 1500 U / ml. Because it is animal-derived, it may contain non-human proteins, which can trigger an immune response; therefore, recombinant human hyaluronidase can be used. As a recombinant human gene, the recombinant human hyaluronidase is a modified form of hyaluronidase, produced in genetically engineered Chinese hamster ovary (CHO) cells, etc. It is soluble and exhibits activity at both neutral and acidic pH levels, thus having a wide range of applications. Furthermore, compared to animal-derived hyaluronidase, it can be prepared at a high activity concentration of over 100,000 U / mg.

[0077] According to another embodiment of the present invention, the present invention provides a nanoparticle comprising sirolimus, albumin and hyaluronidase.

[0078] The nanoparticles of one embodiment of the present invention can be prepared by the above-described method for preparing nanoparticles.

[0079] In one embodiment of the invention, the sirolimus binds to albumin.

[0080] In one embodiment of the invention, the weight ratio of sirolimus to albumin is 1:5 to 1:20, but is not limited thereto.

[0081] In one embodiment of the invention, the nanoparticles further comprise a lyophilization protectant. The lyophilization protectant is as described above.

[0082] In one embodiment of the invention, the nanoparticles can be pharmaceutically formulated for administration to a patient. Therefore, according to another embodiment of the invention, a pharmaceutical composition for treating cancer is provided, comprising the nanoparticles.

[0083] The pharmaceutical composition may be used for the treatment of cancer, with sirolimus as the active ingredient. For example, the cancer may be angioepithelial neoplasia (PEComa), but is not limited thereto.

[0084] Various dosage forms and drug delivery systems available in the technical field to which this invention pertains can be used as dosage forms for the pharmaceutical compositions. For example, see Gennaro, AR, ed. (1995) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.

[0085] In one embodiment of the present invention, the nanoparticles of the present invention, as a pharmaceutical composition, can be administered via one of the following routes: oral; parenteral, for example, percutaneously, intranasally, intramuscularly, intravenously, subcutaneously, intradermally, intratumorally, etc.

[0086] In one embodiment of the present invention, the nanoparticles of the present invention may contain solid, liquid, semi-solid or gaseous excipients.

[0087] The solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, and dried skim milk.

[0088] The liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol, and petrolatum, including a variety of oils derived from animals, plants, or synthetic sources, such as peanut oil, soybean oil, mineral oil, and sesame oil. Preferred liquid carriers, especially for injectable solutions, include water, physiological saline, aqueous dextrose, and glycols. Other suitable pharmaceutical excipients and their dosage forms are described in Remington's Pharmaceutical Sciences, edited by EW Martin (Mack Publishing Company, 18th ed., 1990).

[0089] In a specific embodiment of the present invention, the nanoparticles can be formulated for subcutaneous injection.

[0090] [The effects of the invention]

[0091] The features and advantages of this invention can be summarized as follows:

[0092] (a) This invention provides a method for preparing nanoparticles comprising sirolimus and albumin. (b) This invention provides nanoparticles comprising sirolimus, albumin, and hyaluronidase. (c) This invention provides a pharmaceutical composition comprising said nanoparticles for treating cancer.

[0093] Using the preparation method of the present invention, nanoparticles containing sirolimus and albumin with excellent particle size distribution stability can be prepared. The prepared nanoparticles can be formulated into pharmaceutical compositions suitable for subcutaneous injection, improving the dosage and convenience of administration. Attached Figure Description

[0094] Figure 1 This graph shows the concentration of sirolimus in the blood after intravenous or subcutaneous injection, in order to confirm the pharmacokinetic information of the sirolimus and albumin nanoparticles of the present invention. Detailed Implementation

[0095] The present invention will now be described in more detail through embodiments. However, it will be apparent to those skilled in the art that these embodiments are merely for illustrating the invention more specifically, and the scope of the invention is not limited to these embodiments according to the spirit of the invention.

[0096]

Example

[0097] Unless otherwise specified throughout this specification, the "%" used to indicate the concentration of a particular substance is (weight / )% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0098] [Example 1: Preparation of nanoparticles containing sirolimus and albumin]

[0099] Nanoparticles containing sirolimus and albumin were prepared in the following manner.

[0100] More specifically, 3.17 g of sirolimus was dissolved in chloroform to prepare a 30% w / v sirolimus solution (first solution). A 20% w / v albumin solution was diluted to prepare a 12% albumin solution (second solution, Greencross Human Serum Albumin Inj.). After adding the first solution to the prepared second solution, the mixture was mixed for 5 minutes at 5000–10000 rpm in a high-speed homogenizer (IKA, Germany). The mixture was then transferred to a high-pressure homogenizer and homogenized at 5000–15000 psi for 2–3 cycles. The homogenized mixture was diluted with 6 to 7 times its volume of water.

[0101] Following the same method described above, nanoparticles were prepared by varying the concentration of the first or second solution and the amount of water used in the dilution. The specific variable conditions for different embodiments are shown in Table 1 below.

[0102] Table 1: Preparation methods of nanoparticles containing sirolimus and albumin under different conditions

[0103]

[0104] [Example 2. Particle size tendency of nanoparticles from different processes]

[0105] Dissolve 4.26 g of sirolimus in chloroform to prepare a 44% w / v sirolimus solution (first solution). Dilute a 20% w / v albumin solution to prepare an albumin solution (second solution, 20% Green Cross albumin injection). Add the first solution to the prepared second solution and mix the mixture for 5 minutes at 5000–10000 rpm in a high-speed homogenizer (IKA, Germany). Then, transfer the mixture to a high-pressure homogenizer and homogenize it for 2–3 cycles at 5000–15000 psi. Dilute the homogenized mixture with 6 to 7 times its volume of water.

[0106] After dilution, the organic solvent was removed by vacuum drying using a rotary evaporator under pressure conditions ranging from 20°C to 50°C and 30 hPa to 60 hPa.

[0107] After the dispersion, which has had its organic solvent removed, is filtered through a sterile filter (0.22 μm filter) and dried under reduced pressure, it is filled into vials for freeze drying.

[0108] To confirm the formation and stability of particle size distribution in different processes, the particle size of the mixture after high-pressure homogenization with water dilution, the mixture after vacuum drying, the mixture after aseptic filtration, and the mixture after freeze-drying and rehydration were measured. Particle size was measured using Marlvern's Zetasizer with light scattering (laser angle 90 degrees, temperature 20°C, solvent: distilled water).

[0109] Table 2: Results of different preparation processes for sirolimus and albumin nanoparticles

[0110]

[0111] The particle size measurements confirmed that the particle size increased significantly after aseptic filtration and freeze-drying.

[0112] Then, after rehydrating multiple freeze-dried samples, the strength tendency and deviation between the samples were confirmed, and the measured particle size is shown in Table 3 below.

[0113] Table 3: Particle size results of sirolimus and albumin nanoparticles after freeze-drying

[0114]

[0115] It was confirmed that the particle size of nanoparticles in the freeze-dried and rehydrated liquid increased by approximately 30 nm to 50 nm compared to the liquid that underwent sterile filtration before freeze-drying. Furthermore, discrepancies were also observed between samples that were freeze-dried simultaneously.

[0116] [Example 3. Preparation of sirolimus and albumin nanoparticles with mixed lyophilization protectants]

[0117] The sirolimus solution (first solution) dissolved in chloroform and the albumin solution (second solution) were mixed in a high-speed homogenizer (IKA, Germany) at 5000–10000 rpm for 5 minutes. The mixture was then transferred to a high-pressure homogenizer and homogenized twice at 5000–15000 psi. The homogenized mixture was diluted with water.

[0118] After dilution, the organic solvent is removed by vacuum drying using a rotary evaporator.

[0119] After sterile filtration and vacuum drying to remove the organic solvent dispersion, mannitol, sucrose, and trehalose, which serve as freeze-drying protectants, are dissolved at a concentration of 2% w / v to 10% w / v and then filled into glass vials for freeze-drying.

[0120] The particle size of the liquid undergoing sterile filtration is measured, and the particle size of the freeze-dried sample is measured based on the presence or absence of a freeze-drying protectant.

[0121] Table 4: Particle size results of freeze-dried samples with or without lyophilization protectant.

[0122]

[0123] Measurement results confirmed that, except for 2% concentrations of sucrose and trehalose, no tendency for particle size increase was observed when mixed with freeze-drying protectants.

[0124] [Example 4. Preparation of nanoparticles containing a mixture of lyophilization protectant, sirolimus of hyaluronidase, and albumin]

[0125] Nanoparticles containing sirolimus, albumin, and hyaluronidase were prepared in the following manner.

[0126] More specifically, 3.17 g of sirolimus was dissolved in 4.3% chloroform to prepare a 30% w / v sirolimus solution (first solution). A 20% w / v albumin solution was diluted to prepare a 12% albumin solution (second solution, 20% Green Cross albumin injection). After adding the first solution to the prepared second solution, the mixture was mixed for 5 minutes at 5000–10000 rpm in a high-speed homogenizer (IKA, Germany). The mixture was then transferred to a high-pressure homogenizer and homogenized 2–3 times at 5000–15000 psi. The homogenized mixture was diluted with 6 to 7 times its volume of water.

[0127] After dilution, the organic solvent was removed by vacuum drying using a rotary evaporator under pressure conditions ranging from 20°C to 50°C and 30 hPa to 60 hPa.

[0128] After sterile filtration and vacuum drying to remove the organic solvent from the dispersion, it is dissolved together with lyophilization protectants (mannitol, sucrose, trehalose) at concentrations ranging from 3% w / v to 10% w / v and then filled into vials. Animal-derived hyaluronidase is added to the vials at concentrations of 2000 U / ml (10000 U per vial) and 4000 U / ml (20000 U per vial) before lyophilization.

[0129] [Table 5: Particle size results of freeze-dried samples based on the presence or absence of lyophilization protectant and hyaluronidase]

[0130]

[0131] As shown above, it can be confirmed that without the use of mannitol as a freeze-drying protectant, the particle size increases sharply after freeze-drying. Furthermore, in the case of hyaluronidase, it was confirmed that when the concentration was 4000 U / ml (higher than 2000 U / ml), the particle size remained smaller after freeze-drying.

[0132] [Example 5. Pharmacokinetics of nanoparticles containing sirolimus, hyaluronidase, and albumin, combined with a mixed lyophilization protectant]

[0133] The inventors prepared albumin containing a lyophilization protectant (5% mannitol) and hyaluronidase (0 U / ml, 2000 U / ml, 4000 U / ml) into nanoparticles using the same method as in Example 4, and conducted pharmacokinetic studies based on their route of administration in the following manner.

[0134] Table 6: PK Experimental Information of Sirolimus and Albumin Nanoparticles of the Present Invention

[0135]

[0136] Group 1 consisted of SD rats administered an intravenous injection, while groups 2 to 4 consisted of SD rats administered a subcutaneous injection. Blood samples were collected at different time points to measure the concentration of sirolimus in whole blood. The results are shown in Table 7. Figure 1 As shown.

[0137] Table 7: Pharmacokinetic parameters (mean ± standard deviation) of different groups of sirolimus and albumin nanoparticles of the present invention

[0138]

[0139] As shown in Table 7 and Figure 1 As shown, in terms of relative AUC, for the same dose based on route of administration, G2 (subcutaneous injection) increased drug exposure by 6.5% compared to G1 (intravenous injection). In terms of relative AUC, for the same dose and route of administration based on hyaluronidase addition, G3 (including hyaluronidase) increased drug exposure by 6.8% compared to G2 (without hyaluronidase), and G4 (including a high dose of hyaluronidase) increased it by 13.1% compared to G2. A slightly different trend was observed compared to data from trastuzumab subcutaneous injection (Herceptin hylecta). According to the “NDA / BLA Multi-Disciplinary Review and Evaluation (761106)”, in the case of trastuzumab subcutaneous injection, Tmax was earlier (24-29h vs 67h) compared to the formulation without hyaluronidase, without affecting Cmax or AUC. However, it can be clearly confirmed in the embodiments of the present invention that the Tmax of the nanoparticles of the present invention mixed with hyaluronidase is advanced, and the Cmax and AUC are increased. Compared with intravenous injection (IV), the initial burst release curve is transformed into a slow release curve.

[0140] Therefore, based on these results, the use of hyaluronidase can relatively increase bioavailability compared to the absence of hyaluronidase, and the Cmax is significantly lower compared to the existing IV administration route, thereby reducing adverse drug reactions. Taking these results into account, it is considered an effective subcutaneous injection agent with convenient administration and high patient compliance.

Claims

1. A method for preparing nanoparticles containing sirolimus and albumin, characterized in that, include: Step (a): Prepare a first solution of sirolimus dissolved at a concentration of 5 w / v% to 75 w / v%. Step (b): Prepare a second solution in which albumin is dissolved at a concentration of 5 w / v% to 30 w / v%. Step (c) involves mixing and stirring the first and second solutions at a volume ratio of 1:5 to 1:70; and Step (d) involves diluting the solution, based on the volume of the solution treated in step (c), with 2 to 10 times the volume of an aqueous solvent.

2. The method for preparing nanoparticles containing sirolimus and albumin according to claim 1, characterized in that, It also includes step (e), in which the organic solvent is removed by drying the diluted solution under reduced pressure.

3. The method for preparing nanoparticles containing sirolimus and albumin according to claim 2, characterized in that, It also includes step (f), filtering and drying the solution treated in step (e) to obtain nanoparticles.

4. The method for preparing nanoparticles containing sirolimus and albumin according to claim 3, characterized in that, The drying process is freeze-drying, in which a freeze-drying protectant is added.

5. The method for preparing nanoparticles containing sirolimus and albumin according to claim 4, characterized in that, Hyaluronidase is added during the freeze-drying process.

6. The method for preparing nanoparticles containing sirolimus and albumin according to claim 1, characterized in that, The albumin is selected from the group consisting of human serum albumin, bovine serum albumin, ovalbumin, recombinant human serum albumin, and combinations thereof.

7. The method for preparing nanoparticles containing sirolimus and albumin according to claim 1, characterized in that, The step (c) of mixing and stirring the first solution and the second solution includes a high-pressure homogenization process.

8. The method for preparing nanoparticles containing sirolimus and albumin according to claim 7, characterized in that, The high-pressure homogenization was carried out under pressure conditions ranging from 5,000 psi to 30,000 psi.

9. A nanoparticle, characterized in that, It contains sirolimus, albumin, and hyaluronidase.

10. The nanoparticles according to claim 9, characterized in that, Sirolimus binds electrostatically to albumin.

11. The nanoparticles according to claim 9, characterized in that, The weight ratio of sirolimus to albumin is 1:5 to 1:

20.

12. The nanoparticles according to claim 9, characterized in that, It also contains a freeze-drying protectant.

13. A pharmaceutical composition for treating cancer, characterized in that, It comprises the nanoparticles according to any one of claims 9 to 12.

14. The pharmaceutical composition for treating cancer according to claim 13, characterized in that, The pharmaceutical composition is a subcutaneous dosage form used to treat cancer or as an immunosuppressant.

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