Sirolimus-albumin composition and preparation method therefor

Abstract

The present application discloses a sirolimus-albumin composition and a preparation method therefor. The preparation method of the present application incorporates an incubation step before solvent removal via evaporation of liquid having undergone high-pressure homogenization, thereby resulting in more uniform nanoparticles and further improving the physical stability of a nanosuspension and the chemical stability of a formulation without the need for any stabilizer. Compared with marketed products, the sirolimus-albumin composition of the present application can be completely dissolved within 1-3 min, the reconstitution operation is simpler, and a nanoparticle suspension after reconstitution remains stable at 30°C for 24 h, thereby offering greater convenience for clinical use. In addition, the composition of the present application can be stored at room temperature, is convenient to transport, and has a longer shelf life.

Description

A sirolimus albumin composition and a preparation method thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN2024113566923 filed on September 27, 2024, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of medicine, and specifically relates to a sirolimus albumin composition and a preparation method thereof. BACKGROUND

[0004] Sirolimus, also known as rapamycin, is an mTOR (mammalian target of rapamycin) inhibitor. It inhibits the activation of mTORC1 by binding to FKBP12 (FK506-binding protein-12), thereby inhibiting cytokine-induced T cell activation and proliferation and the progression from the G1 phase to the S phase of the cell cycle. Mechanistically, sirolimus has anti-tumor activity, but its poor water solubility limits its effective delivery and application in the field of tumors.

[0005] The current marketed preparation is sirolimus oral solution / tablets developed by American Wyeth Company, which is used for treating rejection after kidney transplantation, and the trade name is Rapamune. In 2002, it was listed in China. Subsequently, American Pfizer Company developed Temsirolimus (esterification of sirolimus) injection, which is used for treating advanced renal cell carcinoma (Europe and the United States) and mantle cell lymphoma (Europe), and the trade name is Torisel. It has not been marketed in China yet. However, the Temsirolimus injection formula contains surfactants such as Tween and ethanol, and the presence of surfactants may cause irritation, local inflammation and reduce the efficacy of the drug. In order to avoid potential hypersensitivity reactions, drug pretreatment and slow intravenous infusion administration are required.

[0006] Human blood albumin is an endogenous substance in the human body, has good biocompatibility, and can be used as a natural carrier for hydrophobic drugs to increase the solubility of water-insoluble drugs. Abraxis Company in the United States developed Abraxane (albumin-bound paclitaxel) using human blood albumin as an excipient, which was approved by FDA in 2005 and was used for treating breast cancer, non-small cell lung cancer, pancreatic cancer and gastric cancer (Japan) and the like. The company developed sirolimus albumin nanoparticles (Nab-Sirolimus) using the same technology. ​ABI-009) and the clinical research of the product is responsible by AADi company. It has been approved by FDA on November 23, 2021 for the treatment of unresectable locally advanced or metastatic malignant PEComa in adult patients, and has not been approved in China.

[0007] The product is disclosed in the package insert to include 100 mg sirolimus, 850 mg human blood albumin per bottle. The preparation steps of the product in use are complex, 20 ml of normal saline is used to resuspend the freeze-dried powder, the whole dissolution time is at least 8 minutes, if foam or aggregation occurs, at least 15 minutes is needed to increase the foam dissipation, the dispensing time is long, and the clinical use is not convenient. Moreover, the stability of the product is poor, and it needs to be stored at 2° to 8°C, which increases the storage and transportation cost compared with normal temperature storage; the suspension after resuspension should be used immediately in the bottle, if not used immediately, it can be refrigerated at 2°C to 8°C for at most 6 hours; the suspension should be used immediately after being transferred to the infusion bag, if not used immediately, it can be refrigerated at 2°C to 8°C for at most 9 hours; the two processes should not be refrigerated at 2°C to 8°C for more than 15 hours; the room temperature (25°C) can be placed for at most 4 hours in the dark. It can be seen that the product can be placed at room temperature for a short time after resuspension, which is strict in the requirements of clinical dispensing and drug delivery time. In addition, FDA raised in the letter that the product has allergic reactions, which may cause safety problems.

[0008] Therefore, there is a need for a sirolimus albumin composition with simple preparation and good stability to meet the clinical needs. SUMMARY

[0009] The present application provides a sirolimus albumin composition and a preparation method thereof. The sirolimus albumin composition obtained by the preparation method of the present application overcomes the shortcomings of the existing marketed product, has a short dispensing time, simple operation and good stability, and is more suitable for clinical use.

[0010] In a first aspect, the present application provides a preparation method of a sirolimus albumin composition, comprising the following steps:

[0011] (1) Oil phase preparation

[0012] Weigh sirolimus, add solvent, stir until completely dissolved to obtain oil phase solution;

[0013] (2) Water phase preparation

[0014] Weigh albumin, add water for injection, stir to obtain water phase solution;

[0015] (3) Preparation of nanoparticle suspension

[0016] ​The oil phase solution and the water phase solution are mixed and emulsified, high pressure homogenized, incubated, and once evaporated to obtain the nanoparticle suspension, wherein the incubation temperature is 40℃±15℃, preferably 40℃±10℃ or 35-50℃ or 40℃±5℃, and the incubation time is about 0.1-10min, preferably 0.2-8min or 0.3-7min or 0.5-5min or 0.5-3min, preferably 1-2min.

[0017] In some embodiments, the step (1) optionally comprises a filtration step.

[0018] In some embodiments, the step (1) dissolves the sirolimus in the solvent. Suitable solvents include, for example, ketones, esters, ethers, chlorinated solvents, and other solvents known in the art. For example, the solvent is selected from the group consisting of dichloromethane, chloroform (trichloromethane), dichloromethane / ethanol, dichloromethane / tert-butanol, chloroform / ethanol, or chloroform / tert-butanol (e.g., the volume ratio of the mixed solvents is about (1-15):(1-15) or (3-14):(3-14) or (4-13):(4-13) or (5-12):(5-12), preferably (1-15):1 or (3-14):1 or (4-13):1 or (5-12):1, for example, about 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or a range between any two of the foregoing, or about 3:7, 5:7, 4:6, 5:5, 6:5, 8:5, 9:5, 9.5:5, 5:3, 7:3, 6:4, or 9.5:0.5).

[0019] In some embodiments, the present application provides a method for preparing a sirolimus-albumin composition, comprising the following steps:

[0020] (1) Oil phase preparation

[0021] Weigh sirolimus, add dichloromethane, stir until completely dissolved; after dissolving, filter to obtain an oil phase solution;

[0022] (2) Water phase preparation

[0023] Weigh albumin, add water for injection, stir to obtain a water phase solution;

[0024] (3) Preparation of nanoparticle suspension

[0025] The oil phase solution and the aqueous phase solution are mixed and emulsified, high pressure homogenized, incubated, and primary evaporated to obtain the nanoparticle suspension, wherein the incubation temperature is 40°C±15°C, preferably 40°C±10°C or 35-50°C or 40°C±5°C, and the incubation time is about 0.1-10 min, preferably 0.2-8 min or 0.3-7 min or 0.5-5 min or 0.5-3 min, preferably 1-2 min.

[0026] Optionally, the method comprises step (4) secondary evaporation: the nanoparticle suspension obtained in step (3) is subjected to secondary evaporation.

[0027] Optionally, the method comprises step (5) sterilization filtration.

[0028] Optionally, the method comprises step (6) sterile filling.

[0029] Optionally, the method comprises step (7) freeze-drying.

[0030] In some embodiments, the oil phase solution obtained in step (1) contains 100-400 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 100 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 150 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 200 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 250 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 300 mg / ml of sirolimus. In some embodiments, the oil phase solution obtained in step (1) contains 400 mg / ml of sirolimus.

[0031] In some embodiments, the filtration in step (1) is filtration through a 0.2 μm filter cartridge or filter membrane. In some embodiments, the solution obtained after the filtration in step (1) is shaken to obtain the oil phase solution.

[0032] In some embodiments, the albumin in step (2) is albumin of natural origin or synthetically prepared. In some embodiments, the albumin is human albumin or human serum albumin (HSA). In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin in step (2) is preferably human serum albumin, more preferably a human serum albumin solution, which can be a commercially available product, and the concentration is preferably 5-30%, for example 5% or 10% or 15% or 20% or 25% or 30% or a range between any two of the foregoing, wherein the concentration of the human serum albumin solution is weight by volume percentage (w / v). In some embodiments, the albumin in step (2) is a 20% w / v human serum albumin solution.

[0033] In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 10-70 mg / ml or 20-60 mg / ml or 20-40 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 10 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 20 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 30 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 40 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 50 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 60 mg / ml. In some embodiments, the concentration of albumin in the aqueous phase solution resulting from step (2) is 70 mg / ml.

[0034] In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of albumin to sirolimus of (1.5-12): 1 or (2-12): 1 or (2.5-12): 1 or (2.8-12): 1 or (3-12): 1, preferably (2-10): 1 or (3-10): 1 or (3-9): 1 or (3-8): 1 or (3-7): 1 or (3-6): 1 or (3-5): 1 or (3-4): 1, such as 1.5: 1, 1.8: 1, 2: 1, 2.5: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, or a range between any two of the foregoing, wherein the protein drug ratio is by weight, the weight of albumin = the amount of albumin solution fed (v) x the concentration of albumin solution (w / v). In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of (2-10): 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of (3-10): 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of (3-8): 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of (3-5): 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 10: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 7.5: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 5: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 4: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 3: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 2.5: 1. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) at a protein drug ratio of 2: 1.

[0035] In some embodiments, the oil phase solution and the aqueous phase solution are mixed in step (3) by mixing and emulsifying via the oil phase pump and the aqueous phase pump at appropriate speeds through a high shear dispersion emulsifier, passing through a high pressure homogenizer for particle size adjustment, and then passing through an incubator for incubation, and then entering a falling film evaporator for one-time evaporation to remove the solvent to obtain the nanoparticles.

[0036] In some embodiments, the equipment parameters in step (3) are preferably one or more of the following:

[0037] Oil phase pump flow rate 10-20 ml / min, preferably 13-18 ml / min or 14-17 ml / min or 15-17 ml / min or 16-17 ml / min;

[0038] Water phase pump flow rate 200-500 ml / min, preferably 250-450 ml / min or 300-400 ml / min or 350-400 ml / min or 360-380 ml / min;

[0039] Water phase speed is 10-50 times the oil phase speed, preferably 15-30 times or 20-25 times;

[0040] High shear dispersing emulsifier speed 5000 rpm-20000 rpm, preferably 7000 rpm-15000 rpm or 8000 rpm-12000 rpm or 9000 rpm-11000 rpm or 10000 rpm;

[0041] High pressure homogenizer pressure 10000 PSI-25000 PSI, preferably 13000 PSI-22000 PSI or 15000 PSI-20000 PSI or 16000 PSI-20000 PSI or 18000-20000 PSI;

[0042] Falling film evaporator heating temperature 45°C-60°C, preferably 50°C-55°C; initial vacuum control less than 100 mbar, preferably less than 90 mbar or less than 80 mbar.

[0043] In some embodiments, the incubation temperature in step (3) is from 25°C to 55°C. In some embodiments, the incubation temperature in step (3) is from 30°C to 50°C. In some embodiments, the incubation temperature in step (3) is from 35°C to 50°C. In some embodiments, the incubation temperature in step (3) is from 35°C to 45°C. In some embodiments, the incubation temperature in step (3) is from 35°C to 40°C. In some embodiments, the incubation temperature in step (3) is 35°C. In some embodiments, the incubation temperature in step (3) is 40°C.

[0044] In some embodiments, the incubation time in step (3) is 0.5-5 min. In some embodiments, the incubation time in step (3) is 0.5-3 min. In some embodiments, the incubation time in step (3) is 0.5-2.5 min. In some embodiments, the incubation time in step (3) is 1-2 min. In some embodiments, the incubation time in step (3) is 30 s. In some embodiments, the incubation time in step (3) is 60 s. In some embodiments, the incubation time in step (3) is 90 s. In some embodiments, the incubation time in step (3) is 120 s.

[0045] In some embodiments, the incubation in step (3) is for 0.5-5 min at 25-55 °C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 30-50 °C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 30-50 °C. In some embodiments, the incubation in step (3) is for 1-2 min at 30-50 °C. In some embodiments, the incubation in step (3) is for 60 s at 30-50 °C. In some embodiments, the incubation in step (3) is for 90 s at 30-50 °C. In some embodiments, the incubation in step (3) is for 120 s at 30-50 °C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 35-50 °C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 35-50 °C. In some embodiments, the incubation in step (3) is for 1-2 min at 35-50 °C. In some embodiments, the incubation in step (3) is for 60 s at 35-50 °C. In some embodiments, the incubation in step (3) is for 90 s at 35-50 °C. In some embodiments, the incubation in step (3) is for 120 s at 35-50 °C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 35-45 °C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 35-45 °C. In some embodiments, the incubation in step (3) is for 1-2 min at 35-45 °C. In some embodiments, the incubation in step (3) is for 60 s at 35-45 °C. In some embodiments, the incubation in step (3) is for 90 s at 35-45 °C. In some embodiments, the incubation in step (3) is for 120 s at 35-45 °C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 35-40 °C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 35-40 °C. In some embodiments, the incubation in step (3) is for 1-2 min at 35-40 °C. In some embodiments, the incubation in step (3) is for 60 s at 35-40 °C. In some embodiments, the incubation in step (3) is for 90 s at 35-40 °C. In some embodiments, the incubation in step (3) is for 120 s at 35-40 °C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 35 °C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 35 °C. In some embodiments, the incubation in step (3) is for 1-2 min at 35 °C.In some embodiments, the incubation in step (3) is for 60 s at 35°C. In some embodiments, the incubation in step (3) is for 90 s at 35°C. In some embodiments, the incubation in step (3) is for 120 s at 35°C. In some embodiments, the incubation in step (3) is for 0.5-5 min at 40°C. In some embodiments, the incubation in step (3) is for 0.5-3 min at 40°C. In some embodiments, the incubation in step (3) is for 1-2 min at 40°C. In some embodiments, the incubation in step (3) is for 60 s at 40°C. In some embodiments, the incubation in step (3) is for 90 s at 40°C. In some embodiments, the incubation in step (3) is for 120 s at 40°C.

[0046] In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 30-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 30-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 30-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 30-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 30-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 30-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 30-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 30-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 30-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 30-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 30-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 30-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 35-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 35-50 °C for 0.5-3 min.In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (2-10): 1 in step (3), and the incubation is at 35-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 35-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 35-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-10): 1 in step (3), and the incubation is at 35-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 35-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 35-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-8): 1 in step (3), and the incubation is at 35-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 35-50 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 35-50 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of (3-5): 1 in step (3), and the incubation is at 35-50 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of 4: 1 in step (3), and the incubation is at 35-40 °C for 0.5-5 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of 4: 1 in step (3), and the incubation is at 35-40 °C for 0.5-3 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of 4: 1 in step (3), and the incubation is at 35-40 °C for 1-2 min. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of 4: 1 in step (3), and the incubation is at 35 °C for 90 s. In some embodiments, the oil phase solution and the aqueous phase solution are mixed in a protein drug ratio of 4: 1 in step (3), and the incubation is at 40 °C for 90 s.

[0047] In some embodiments, the secondary evaporation in step (4) is performed using a falling film evaporator.

[0048] In some embodiments, the falling film evaporator used in the secondary evaporation in step (4) has a heating temperature of 45-60°C, preferably 50-55°C; and an initial vacuum control of less than 100 mbar, preferably less than 90 mbar or less than 80 mbar.

[0049] In the above steps (3) and (4), the emulsification time, the high pressure homogenization time, the evaporation time, etc. are related to the volume of the feed liquid, and those skilled in the art are able to determine and adjust the above time parameters according to actual needs based on the disclosure of the present application.

[0050] In the preparation method of the present application, steps (5), (6) and (7) are performed in a conventional manner known to those skilled in the art.

[0051] Those skilled in the art can understand that the above embodiments can be combined in any manner, and the technical solutions thus obtained and their modifications are also included in the scope of the present application.

[0052] In a second aspect, the present application provides a sirolimus albumin composition obtained by the preparation method of the first aspect described above.

[0053] In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is (1.5-12): 1 or (2-12): 1 or (2.5-12): 1 or (2.8-12): 1 or (3-12): 1, preferably (2-10): 1 or (3-10): 1 or (3-9): 1 or (3-8): 1 or (3-7): 1 or (3-6): 1 or (3-5): 1 or (3-4): 1, for example 1.5: 1, 1.8: 1, 2: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or a range between any two of the foregoing. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is (2-10): 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is (3-10): 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is (3-8): 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is (3-5): 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 10: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 8: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 5: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 4: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 3: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 2.5: 1. In some embodiments, the weight ratio of albumin to sirolimus in the sirolimus albumin composition is 2: 1.

[0054] In some embodiments, the sirolimus albumin composition is obtained by steps (1)-(3) or steps (1)-(6) of the preparation method of the first aspect described above, and the composition is in the form of a nanoparticle suspension.

[0055] In some embodiments, the sirolimus albumin composition is obtained by steps (1)-(7) of the preparation method of the first aspect described above, and the composition is in the form of a lyophilized powder.

[0056] In some embodiments, the sirolimus albumin composition is in the form of a nanoparticle suspension, wherein the average particle size of the nanoparticles is 50-200 nm, preferably 80-150 nm or 85-145 nm or 90-140 nm, more preferably 90-130 nm or 90-120 nm, and / or the polydispersity index (PDI) is <0.5, preferably <0.4, more preferably <0.3 or <0.25 or <0.2 or <0.18 or <0.15.

[0057] In some embodiments, the sirolimus albumin composition is in the form of a lyophilized powder, which can be completely dispersed within 5 min, preferably within 4 min, 3 min or 2 min, more preferably within 1 min.

[0058] In some embodiments, the sirolimus albumin composition can be stored at 2-8°C for more than 24 months.

[0059] In some embodiments, the sirolimus albumin composition can be stored at room temperature.

[0060] In some embodiments, the sirolimus albumin composition can be stored at 30°C, relative humidity 65.0% for more than 18 months.

[0061] In some embodiments, the sirolimus albumin composition is a lyophilized powder, which can be reconstituted with 5% glucose injection or 0.9% sodium chloride injection, preferably with 5% glucose injection.

[0062] In some embodiments, the sirolimus albumin composition is a lyophilized powder, which can be stored at 2-8°C for more than 15 hours, for example, for 24 hours after reconstitution.

[0063] In some embodiments, the sirolimus albumin composition is a lyophilized powder, which can be stored at 30°C for 24 hours after reconstitution.

[0064] In some embodiments, the sirolimus albumin composition does not contain a stabilizer.

[0065] In a third aspect, the present application provides use of the sirolimus albumin composition of the second aspect described above in the preparation of a medicament for treating tumors, mitochondrial-related disorders, central nervous system diseases, pulmonary arterial hypertension.

[0066] In a fourth aspect, the present application provides a method for treating tumors, mitochondrial-related disorders, central nervous system diseases or pulmonary arterial hypertension, the method comprising: administering the sirolimus albumin composition of the second aspect described above to a patient or subject in need of treatment.

[0067] In a fifth aspect, the present application provides the sirolimus albumin composition of the second aspect described above for use in treating a tumor, a mitochondria-related disorder, a central nervous system disease, or pulmonary arterial hypertension.

[0068] In some embodiments, the tumor described herein is selected from liver cancer, lung cancer, pancreatic cancer, renal cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, head and neck cancer, bladder cancer, malignant perivascular epithelioid cell tumor (PEComa), mantle cell lymphoma, multiple myeloma, lymphangioleiomyomatosis, Hodgkin lymphoma.

[0069] The term

[0070] As used herein, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a partial or complete resolution of a disease state, reducing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, improving quality of life, and / or prolonging survival. In some embodiments, treatment reduces the severity of one or more symptoms associated with cancer by at least any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to the corresponding symptoms in the same subject prior to treatment or as compared to the corresponding symptoms in other subjects who did not receive the treatment. "Treatment" also includes reducing the pathological consequences of a disease. The methods of the present application contemplate any one or more of these aspects of treatment.

[0071] As used herein, the term "mitochondrial-associated disorder" refers to any disease or disorder caused by dysfunction of mitochondria. Mitochondrial-associated disorders can cause a complex array of symptoms. Symptoms of mitochondrial-associated disorders include, for example, muscle weakness, muscle cramps, seizures, food regurgitation, learning disabilities, deafness, short stature, ophthalmoplegia, diabetes, heart problems, and stroke-like episodes. The severity of symptoms of mitochondrial-associated disorders ranges from life-threatening to almost imperceptible. Examples of mitochondrial-associated disorders include, but are not limited to: Leigh syndrome; MELAS syndrome; NARP syndrome; myoclonic epilepsy with ragged red fibers (MERFF); chronic progressive external ophthalmoplegia (CPEO); Kearns-Sayre syndrome (KSS); mitochondrial neurogastrointestinal encephalomyopathy (MNGIE); Friedreich's ataxia; amyotrophic lateral sclerosis (ALS); Huntington's disease; Parkinson's disease; macular degeneration; epilepsy; Alzheimer's disease; Leber's hereditary optic neuropathy (LHON); progressive external ophthalmoplegia (PEO); diabetes; Pearson syndrome; Alper's disease (progressive infantile poliodystrophy); ataxia neuropathy spectrum; autism spectrum disorder; Barth syndrome (fatal infantile cardiomyopathy); carnitine-acyl-carnitine translocase deficiency; carnitine deficiency; carnitine palmitoyltransferase I deficiency; carnitine palmitoyltransferase II deficiency; carnitine-acylcarnitine translocase deficiency; coenzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV / COX deficiency; complex V deficiency; infantile myopathy and lactic acidosis; leukodystrophy with brain stem and spinal cord involvement and lactic acidosis (LBSL); long-chain 3-hydroxyacyl CoA dehydrogenase deficiency (LCHAD); long-chain acyl CoA dehydrogenase deficiency (LCAD); Luft disease; medium-chain acyl CoA dehydrogenase deficiency (MCAD); mtDNA depletion syndrome (MDS); multiple acyl CoA dehydrogenase deficiency (MADD); myoclonic epilepsy myopathy sensory ataxic (MEMSA); pyruvate carboxylase deficiency; pyruvate dehydrogenase complex deficiency (PDCD); short-chain acyl CoA dehydrogenase deficiency (SCAD); and very long-chain acyl CoA dehydrogenase deficiency (VLCAD).

[0072] Central nervous system diseases, also called central nervous system disorders, are a group of neurological disorders that affect the structure or function of the brain or spinal cord, which collectively form the central nervous system (CNS). In some embodiments, the CNS disorder is a glioma. In some embodiments, the CNS disorder is a malignant glioma. In some embodiments, the CNS disorder is epilepsy. In some embodiments, the CNS disorder is a cortical dysplasia (e.g., focal cortical dysplasia). In some embodiments, the CNS disorder is selected from the group consisting of tuberous sclerosis, brain tumor, fragile X syndrome, Down syndrome, Rett syndrome, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.

[0073] Pulmonary hypertension (PH) is a syndrome characterized by elevated pulmonary arterial pressure. PH is defined hemodynamically as a systolic pulmonary arterial pressure greater than 30 mm Hg or an estimated mean pulmonary arterial pressure greater than 25 mm Hg. In some embodiments, the pulmonary hypertension is any one of pulmonary arterial hypertension (PAH), idiopathic pulmonary arterial hypertension (IPAH), heritable pulmonary arterial hypertension (HPAH), drug and toxin-induced PAH, PAH associated with connective tissue disorders, and PAH associated with congenital heart defects. In some embodiments, the pulmonary hypertension is severe pulmonary hypertension. In some embodiments, the pulmonary hypertension is World Health Organization (WHO) functional class II, III, or IV pulmonary arterial hypertension. In some embodiments, the pulmonary hypertension is WHO functional class II pulmonary arterial hypertension. In some embodiments, the pulmonary hypertension is WHO functional class III pulmonary arterial hypertension. In some embodiments, the pulmonary hypertension is WHO functional class IV pulmonary arterial hypertension.

[0074] As used herein, the term “dispersion complete” refers to a suspension obtained after re-suspension of a lyophilized powder with a solvent that is milky and uniform with no visible particles or precipitates.

[0075] As used herein, the term “room temperature” refers to 25 °C ± 5 °C. In some embodiments, room temperature refers to 25 °C.

[0076] Unless otherwise specified, the percentages and parts described in the present application are calculated by weight.

[0077] Unless otherwise specified, the standing described in the present application is standing at room temperature environment.

[0078] For the purpose of providing a more concise description, some quantitative data herein are not expressed with the term "about". It is to be understood that, whether or not explicitly stated, each numerical value given herein is intended to include both the actual value (the given value) and the approximate value (the given value plus or minus a reasonable amount that is within the ordinary skill of the person of ordinary skill in the art). The approximate value is preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, ±1% of the given value. In some embodiments, the approximate value is obtained by rounding.

[0079] The marketed sirolimus albumin nanoparticle preparation The preparation procedure is complicated, and the chemical stability is poor, and it needs to be stored at 2-8°C, and the suspension after resuspension can be placed in the dark at room temperature for at most 4 hours, which is strict for clinical dispensing and dosing time.

[0080] To solve the above problems, the present inventors tried various means in the development process, such as adjusting the protein drug ratio, adjusting the process prescription of the oil phase solution and the water phase solution, etc., to improve the stability of the drug, but a good solution was not obtained. The present inventors unexpectedly found that, after high-pressure homogenization, the addition of an incubation step before evaporation of the solvent such as dichloromethane from the feed liquid not only makes the nanoparticles more uniform, but also further improves the physical stability of the nanoparticle suspension and the chemical stability of the preparation without adding any stabilizer, and even improves the resuspension effect. These effects are beyond the expectations of those skilled in the art.

[0081] Compared with the marketed product The sirolimus albumin composition prepared by the method of the present application has one or more of the following advantages:

[0082] (1) Fast dissolution speed, short preparation time.

[0083] The preparation procedure is complicated, and the chemical stability is poor, and it needs to be stored at 2-8°C, and the suspension after resuspension can be placed in the dark at room temperature for at most 4 hours, which is strict for clinical dispensing and dosing time.

[0084] The sirolimus albumin composition described in the present application can be completely dissolved in 1-3 min, the preparation time is greatly shortened, the operation is more simple, and it is convenient for clinical use.

[0085] (2) Good stability.

[0086] The stability of the suspension is poor, and the resuspended suspension can be placed in the dark at room temperature (25°C) for at most 4 hours, and can be stored in the cold at 2-8°C for at most 15 hours, which is strict for clinical dispensing and administration.

[0087] The sirolimus albumin composition described in the present application has good stability, and the stable time after reconstitution is longer. The sirolimus albumin composition can be stored in the cold at 2-8°C for more than 15 hours, for example, for 24 hours, and can be stable at 30°C for 24 hours whether it is placed in an infusion bottle (glass or polypropylene) or an infusion bag. The better stability provides sufficient time for clinical dispensing and administration, and greatly improves the convenience and safety of clinical use.

[0088] (3) Convenient storage.

[0089] The sirolimus albumin composition needs to be stored at 2-8°C, which increases the storage and transportation costs compared with normal temperature storage.

[0090] The sirolimus albumin composition described in the present application has a milder storage condition, can be stored at normal temperature, is convenient for transportation, and has a longer shelf life. DETAILED DESCRIPTION

[0091] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are only used for demonstration.

[0093] Example 1 Preparation of sirolimus albumin composition

[0094] According to the following preparation method, the sirolimus albumin composition is prepared.

[0095] 1. Preparation prescription

[0096] Table 1 Product prescription composition Note: The dosage of 20% w / v human albumin solution is used as the dosage, and the dosage of albumin = dosage of human albumin solution (v) x 20%. The density of 20% w / v human albumin solution is about 1.04 g / ml.

[0097] 2. Preparation method

[0098] (1) Oil phase preparation

[0099] Siroliumus was weighed, added into dichloromethane, and stirred until completely dissolved; after dissolution, the solution was filtered through a 0.2 μm filter, and shaken to obtain an oil phase solution;

[0100] (2) Preparation of the water phase

[0101] A 20% w / v human serum albumin solution was weighed into the water phase tank, and water for injection was added, and shaken to obtain a water phase solution;

[0102] (3) Preparation of the nanoparticle suspension

[0103] The oil phase solution and the water phase solution were mixed and emulsified by a high shear dispersion emulsifier at an appropriate speed via an oil phase pump and a water phase pump, respectively, and then passed through a high pressure homogenizer for particle size adjustment, and then passed through an incubator for incubation, and then passed through a falling film evaporator for removal of dichloromethane by primary evaporation to obtain a nanoparticle suspension;

[0104] The parameters of the respective devices were as follows: oil phase pump flow rate 16-17 ml / min; water phase pump flow rate 350-380 ml / min, the water phase speed being about 20 times that of the oil phase; high shear dispersion emulsifier speed 10,000 rpm; high pressure homogenizer pressure adjusted to 20,000 PSI; incubator temperature 40°C, incubation time about 90 s; primary evaporation falling film evaporator heating temperature 50°C, initial vacuum control less than 80 mbar;

[0105] (4) Secondary evaporation

[0106] The nanoparticle suspension obtained in step (3) was subjected to secondary evaporation to further remove dichloromethane. The falling film evaporator heating temperature was 50°C, and the initial vacuum control was less than 80 mbar;

[0107] (5) Bacterial removal filtration

[0108] (6) Sterile filling

[0109] (7) Freeze-drying.

[0110] Effect of the process of Example 2 on the properties of the nanoparticle suspension

[0111] Prescription 2-1: Siroliumus 3.02 g was weighed into a conical flask, 15 ml of dichloromethane was added, and an oil phase solution with a concentration of 200 mg / ml was prepared, and filtered for use. A 20% w / v human serum albumin solution 104 g (protein / drug (w / w) = 6.6:1) was weighed, and water for injection was added to make up to 500 ml, and a water phase solution with a concentration of 40 mg / ml was prepared. The oil phase solution was poured into the water phase solution, emulsified, and high pressure homogenized. The homogenized solution was divided into three portions, and the subsequent operations were carried out according to processes 1-3 in Table 2 below. After evaporation, the solution was filtered using a 0.2 μm cellulose acetate membrane, and the drug loading, average particle size, polydispersity index (PDI), and suspension stability of the sample were detected.

[0112] Prescription 2-2: 4.5 g of sirolimus was weighed into a conical flask, 22.5 ml of dichloromethane was added to prepare an oil phase solution with a concentration of 200 mg / ml, and filtration was performed for standby. 198.9 g of 20% w / v human serum albumin solution (protein / drug (w / w) = 8.5:1) was weighed and diluted to 500 ml with water for injection to prepare an aqueous phase solution with a concentration of 76.5 mg / ml. The oil phase was poured into the aqueous phase solution, emulsified, and high-pressure homogenized. The homogenized solution was divided into two parts, and subsequent operations were performed according to the following processes. After evaporation, the solution was filtered through a 0.2 μm cellulose acetate membrane, and the drug loading, average particle size, polydispersity index (PDI), and suspension stability of the sample were detected.

[0113] Table 2 Effect of process on the properties of nanoparticle suspension Note: The drug loading is the mass of drug in the filtered solution per unit area of the filter membrane. Drug loading = drug concentration after filtration x volume of the filtered solution ÷ area of the filter membrane.

[0114] As can be seen by comparing process 1 and 2 in prescription 2-1, increasing the incubation step increases the average particle size from 93 nm to 122 nm, reduces the PDI, improves the stability of the nanoparticles, and significantly increases the drug loading, indicating that incubation can make the nanoparticles more uniform.

[0115] The inventors originally believed that the increase in the suspension stability of the nanoparticles in process 1 was due to the increase in the particle size, but were surprised to find that, by comparing process 1 and 3, although other process methods can also obtain particles with an average particle size of about 120 nm, the PDI and drug loading of process 3 do not improve, and the stability of the particles is poor, and the sample precipitates after 6 h of standing.

[0116] The inventors adjusted the protein / drug ratio to prepare samples according to prescription 2-2, and compared the results of process 2 and process 4, which also indicated that incubation can make the nanoparticles more uniform, increase the drug loading, and improve the stability of the particles.

[0117] The above research results show that incubation is very important in this process, greatly improving the uniformity, suspension stability, and drug loading of the nanoparticle suspension.

[0118] Example 3 Effect of incubation temperature on the properties of nanoparticle suspension

[0119] Referring to the prescription 1-3 in Example 1 and the preparation method thereof, the oil phase solution and the water phase solution were prepared respectively, the concentration of sirolimus in the oil phase solution was 200 mg / mL, the concentration of albumin in the water phase solution was 40 mg / mL, the oil phase solution and the water phase solution were mixed according to the weight ratio of protein to drug of 4:1, and the nanoparticle suspension was prepared by using the method of "oil-water phase mixing emulsification-high pressure homogenization-incubation-one evaporation", the incubation time was fixed at 90 s, and the influence of incubation temperature on the performance of the nanoparticle suspension was compared.

[0120] Table 3 Influence of incubation temperature on the performance of the nanoparticle suspension

[0121] As shown in Table 3, under the above conditions, when the incubation temperature was increased from 25℃ to 40℃, the average particle size of the nanoparticles in the suspension increased from 79 nm to 108 nm, the PDI decreased from 0.368 to 0.124, and the drug overloading amount increased from 15.21 mg / cm 2 to 30.53 mg / cm 2 ; when the incubation temperature was increased from 40℃ to 55℃, the average particle size of the nanoparticles in the suspension increased from 108 nm to 175 nm, and the drug overloading amount decreased from 30.53 mg / cm 2 to 10.51 mg / cm 2 . The above results show that the incubation temperature can affect the performance of the nanoparticle suspension, and appropriately increasing the incubation temperature can make the particles more uniform, but too high incubation temperature can cause the particles to aggregate, resulting in decreased stability.

[0122] Example 4 Influence of incubation time on the performance of the nanoparticle suspension

[0123] Referring to the preparation method of prescription 1-3 in Example 1, the oil phase solution and the water phase solution were prepared respectively, the concentration of sirolimus in the oil phase solution was 200 mg / mL, the concentration of albumin in the water phase solution was 40 mg / mL, the oil phase solution and the water phase solution were mixed according to the weight ratio of protein to drug of 4:1, and the nanoparticle suspension was prepared by using the method of "oil-water phase mixing emulsification-high pressure homogenization-incubation-one evaporation", the incubation temperature was set at 40℃, and the influence of incubation time on the performance of the nanoparticle suspension was compared.

[0124] Table 4 Influence of incubation time on the performance of the nanoparticle suspension

[0125] As shown in Table 4, under the above experimental conditions, the average particle size of the nanoparticles increased with the prolongation of the incubation time. When the incubation time was increased from 30 s to 90 s, the drug overloading amount increased from 14.51 mg / cm 2 to 30.53 mg / cm 2 ; when the incubation time was increased from 90 s to 150 s, the drug overloading amount of the suspension decreased from 30.53 mg / cm2 decreased to 15.03 mg / cm 2 The above results show that the incubation time can affect the performance of the nanoparticle suspension, and too long incubation time can lead to the increase or aggregation of the suspension particles, thereby affecting the drug loading and suspension stability.

[0126] Example 5 Influence of different protein-drug ratios on the performance of nanoparticle suspensions

[0127] Referring to the preparation method described in Example 1, oil phase solution and water phase solution were prepared according to Table 5, respectively, and mixed according to the protein-drug ratio. The nanoparticle suspension was prepared by "oil-water phase mixing-emulsification-high pressure homogenization-incubation-one evaporation". The incubation temperature was 40°C, and the incubation time was 90s. The influence of different protein-drug ratios on the performance of nanoparticle suspensions was compared.

[0128] The results are shown in Table 5. As can be seen from Table 5, by adjusting the oil phase solution sirolimus concentration and water phase solution protein concentration of the process prescription, formulations with different protein-drug ratios are obtained. When the protein-drug ratio is greater than 15, the drug loading is low, and the reconstitution time after lyophilization is longer. When the protein-drug ratio is less than 2.5, the suspension stability of the nanoparticle decreases.

[0129] Example 6 Influence of solvent composition on the performance of nanoparticle suspensions

[0130] Prescription: 1.2g of sirolimus was weighed and added to 12ml of solvent (the solvent composition is shown in Table 6), and stirred until completely dissolved to prepare an oil phase solution with a drug concentration of 100mg / ml. 50g of 20%w / v human serum albumin solution was weighed and added to 270g of water for injection, and shaken to prepare a water phase solution with a protein concentration of 30mg / ml.

[0131] Process 6-1: The oil phase solution was poured into the water phase solution, emulsified, and high-pressure homogenized. After homogenization, the material liquid was incubated in a 40°C incubator for 90s and then evaporated in an evaporator. After evaporation, the material liquid was filtered using a 0.2μm cellulose acetate membrane, and the average particle size, polydispersity index (PDI) and suspension stability of the sample were detected.

[0132] Process 6-2: The oil phase solution was poured into the water phase solution, emulsified, and high-pressure homogenized. After homogenization, the material liquid was directly evaporated in an evaporator. After evaporation, the material liquid was filtered using a 0.2μm cellulose acetate membrane, and the average particle size, polydispersity index (PDI) and suspension stability of the sample were detected.

[0133] Table 6 Influence of solvent composition on the performance of nanoparticle suspensions

[0134] As can be seen from Table 6, under different solvent compositions, the sample with an incubation step has a higher particle size and a lower PDI, and the nanoparticle is more stable.

[0135] Stability test of lyophilized samples of Test Example 1

[0136] The lyophilized samples obtained in Example 1, Formulations 1-3, were used to investigate the stability of the formulations and compared with the marketed product . The formulations of the present application were reconstituted with 5% glucose injection, and the reference product was reconstituted with 0.9% sodium chloride injection according to the product specification.

[0137] The results are shown in Table 7. As shown in Table 7, the content of polymers was equivalent and the content of broken sirolimus was lower (0.63% vs 0.87%) when the product of the present application was stored for 24 months under refrigerated (2-8°C) conditions compared with storage for 17 months. Furthermore, the content of polymers of the product of the present application was equivalent and the content of broken sirolimus was still within the quality control standard (not more than 3%) when the product was stored for 18 months at 30°C and 65.0% relative humidity, which was not significantly different from storage for 17 months under refrigerated conditions. The above results show that the product of the present application has good stability and can be stored at room temperature.

[0138] Table 7. Stability comparison of the product of the present application and Note: The product of the present application contains 50 mg sirolimus and about 200 mg human serum albumin per vial. The storage time is the time from the production date to the test date.

[0139] The commercially available product contains 100 mg sirolimus and about 850 mg human serum albumin per vial. The storage time is the time from the production date indicated in the product specification to the test date.

[0140] The content of polymers (%) was determined by molecular exclusion chromatography and the content of broken sirolimus (%) was determined by high performance liquid chromatography, both of which were calculated by area normalization.

[0141] Reconstitution time of lyophilized samples and stability of reconstituted suspensions of Test Example 2

[0142] According to Formulations 1-3 and the preparation method of Example 1, oil phase solution and water phase solution were prepared, the concentration of sirolimus in the oil phase solution was 200 mg / mL, the concentration of albumin in the water phase solution was 40 mg / mL, the oil phase solution and the water phase solution were mixed at a protein-drug weight ratio of 4:1, and nanoparticles were prepared by “oil-water phase mixing-emulsification-high pressure homogenization-incubation-one evaporation”, the incubation temperature was 40°C, and the incubation time was 90 s. The lyophilized samples were used to investigate the reconstitution time and the stability of the reconstituted suspensions.

[0143] ​The product of the present application was added to the reconstituting solvent and immediately shaken gently with reference to Table 8. The marketed product was reconstituted according to its product specification, i.e. slowly added to the reconstituting solvent for 1 minute, left to stand for 5 minutes, and then gently rotated the vial for 2 minutes until completely dissolved. The concentration of the suspension after reconstitution was 5 mg / ml (calculated as sirolimus).

[0144] As shown in Table 8, the product of the present application was completely dispersed within 1 minute, while the marketed product required 8 minutes. The average particle size and the content of broken sirolimus of the solution of the product of the present application after reconstitution did not change substantially during storage, while the particle size and the content of broken sirolimus of the solution after reconstitution according to the product specification of the marketed product showed a significant increase during storage.

[0145] In addition, the stability of the product of the present application after reconstitution and storage at 30°C for 24 hours was also investigated, and the results are shown in Table 9. As shown in Table 9, the sample of the product of the present application mixed with 5% glucose injection, whether stored in an infusion bottle (glass or polypropylene) or an infusion bag, showed no significant change in the content of sirolimus, the content of polymers, and the average particle size after storage at 30°C for 24 hours, and the content of broken sirolimus met the requirements of the quality standard (less than 3%). However, the solution after reconstitution was stored in an infusion bag at room temperature (25°C) in the dark for a maximum of 4 hours. The above results show that the product of the present application is more stable than the marketed product after reconstitution.

[0146] Table 8 Comparison of the reconstitution time and the stability of the suspension of the product of the present application and the marketed product Note: Cold storage refers to 2-8°C.

[0147] Table 9 Test results of the product of the present application mixed with 5% glucose Note: The concentration of the suspension after reconstitution was 5 mg / ml (calculated as sirolimus).

Claims

1. A method for preparing a sirolimus-albumin composition, comprising the following steps: (1) oil phase preparation weighing sirolimus, adding solvent, stirring until completely dissolved to obtain an oil phase solution; (2) water phase preparation weighing albumin, adding water for injection, stirring to obtain a water phase solution; (3) preparing nanoparticle suspension mixing and emulsifying the oil phase solution and the water phase solution, high pressure homogenization, incubation, one-time evaporation to obtain a nanoparticle suspension, wherein the incubation temperature is 40℃±15℃, preferably 40℃±10℃ or 40℃±8℃ or 35℃-50℃ or 40℃±5℃, the incubation time is about 0.1-10min, preferably 0.2-8min or 0.3-7min or 0.5-5min or 0.5-3min, preferably 1-2min; Optionally, the method comprises step (4) secondary evaporation: the nanoparticle suspension obtained in step (3) is subjected to secondary evaporation; Optionally, the method comprises step (5) sterilization filtration; Optionally, the method comprises step (6) sterile filling; Optionally, the method comprises step (7) freeze-drying.

2. The method for preparing according to claim 1, comprising one or more of the following features: step (1) comprises a filtration step; the solvent in step (1) is selected from ketones, esters, ethers, chlorinated solvents, for example, selected from dichloromethane, chloroform, dichloromethane / ethanol, dichloromethane / tert-butanol, chloroform / ethanol or chloroform / tert-butanol; the oil phase solution obtained in step (1) contains 100mg / ml-400mg / ml of sirolimus; the filtration in step (1) is filtration through a 0.2μm filter cartridge or filter membrane; the albumin in step (2) is naturally derived or synthetically prepared albumin; preferably, the albumin is human albumin or human serum albumin (HSA) or recombinant albumin; the concentration of albumin in the water phase solution obtained in step (2) is 10mg / ml-70mg / ml or 20mg / ml-60mg / ml or 20mg / ml-40mg / ml; the one-time evaporation in step (3) and / or the secondary evaporation in step (4) uses a falling film evaporator for evaporation.

3. The method for preparing according to claim 1 or 2, wherein the albumin in step (2) is preferably human serum albumin, more preferably a human serum albumin solution, wherein the concentration of the human serum albumin solution is preferably 10%-30%, for example 10% or 15% or 20% or 25% or 30% or a range between any two of the foregoing, the concentration of the human serum albumin solution being weight percent by volume.

4. The preparation method of any one of claims 1-3, wherein in step (3), the oil phase solution and the water phase solution are mixed at a certain albumin to sirolimus protein drug ratio, which is (1.5-12): 1 or (2-12): 1 or (2.5-12): 1 or (2.8-12): 1 or (3-12): 1, preferably (2-10): 1 or (3-10): 1 or (3-9): 1 or (3-8): 1 or (3-7): 1 or (3-6): 1 or (3-5): 1 or (3-4): 1, such as 1.5: 1, 1.8: 1, 2: 1, 2.5: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or a range between any two of the foregoing; the protein drug ratio is by weight; the weight of albumin = the feeding amount (v) of albumin solution x the concentration (w / v) of albumin solution.

5. The preparation method of any one of claims 1-4, wherein in step (3), the oil phase solution and the water phase solution are mixed and emulsified by a high shear dispersion emulsifier via an oil phase pump and a water phase pump at appropriate speeds, are granulated by a high pressure homogenizer, are incubated by an incubator, and then are subjected to one-time evaporation to remove the solvent by a falling film evaporator to obtain a nanoparticle suspension; Preferably, the equipment parameters in step (3) are selected from one or more of the following: the flow rate of the oil phase pump is 10 ml / min-20 ml / min, preferably 13 ml / min-18 ml / min or 14 ml / min-17 ml / min or 15 ml / min-17 ml / min or 16 ml / min-17 ml / min; the flow rate of the water phase pump is 200 ml / min-500 ml / min, preferably 250 ml / min-450 ml / min or 300 ml / min-400 ml / min or 350 ml / min-400 ml / min or 360 ml / min-380 ml / min; the speed of the water phase is 10-50 times, preferably 15-30 times or 20-25 times, the speed of the oil phase; the speed of the high shear dispersion emulsifier is 5000 rpm-20000 rpm, preferably 7000 rpm-15000 rpm or 8000 rpm-12000 rpm or 9000 rpm-11000 rpm or 10000 rpm; the pressure of the high pressure homogenizer is 10000 PSI-25000 PSI, preferably 13000 PSI-22000 PSI or 15000 PSI-20000 PSI or 16000 PSI-20000 PSI or 18000-20000 PSI; the heating temperature of the falling film evaporator is 45℃-60℃, preferably 50℃-55℃; the initial vacuum control is less than 100 mbar, preferably less than 90 mbar or less than 80 mbar.

6. The preparation method of claim 2, wherein the falling film evaporator heating temperature of the secondary evaporation in step (4) is 45-60°C, preferably 50-55°C; the initial vacuum control is less than 100 mbar, preferably less than 90 mbar or less than 80 mbar.

7. A sirolimus-albumin composition prepared by the method of any one of claims 1-6.

8. The sirolimus-albumin composition of claim 7, wherein the weight ratio of albumin to sirolimus is (1.5-12): 1 or (2-12): 1 or (2.5-12): 1 or (2.8-12): 1 or (3-12): 1, preferably (2-10): 1 or (3-10): 1 or (3-9): 1 or (3-8): 1 or (3-7): 1 or (3-6): 1 or (3-5): 1 or (3-4): 1, for example 1.5: 1, 1.8: 1, 2: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 or a range between any two of the foregoing.

9. The sirolimus-albumin composition of claim 7 or 8, which is in the form of a nanoparticle suspension, wherein the average particle size of the nanoparticles is 50-200 nm, preferably 80-150 nm or 85-145 nm or 90-140 nm, more preferably 90-130 nm or 90-120 nm, and / or the polydispersity index (PDI) is <0.5, preferably <0.4, more preferably <0.3 or <0.25 or <0.2 or <0.18 or <0.

15.

10. The sirolimus-albumin composition of claim 7 or 8, which is stable at 2-8°C for more than 24 months, is stable at room temperature, or is stable at 30°C, relative humidity 65.0% for more than 18 months.

11. The sirolimus-albumin composition of claim 7 or 8, which is in the form of a lyophilized powder, which is completely dispersed within 5 min, preferably within 4 min, 3 min or 2 min, more preferably within 1 min.

12. The sirolimus-albumin composition of claim 11, wherein the completely dispersed reconstituted suspension is stable at 2-8°C for more than 15 hours or at 30°C for 24 hours.

13. The sirolimus-albumin composition of any one of claims 7-12 for use in the treatment of tumors, mitochondrial-associated disorders, central nervous system diseases, pulmonary arterial hypertension.

14. Use of the sirolimus-albumin composition of any one of claims 7-12 for the preparation of a medicament for the treatment of tumors, mitochondrial-associated disorders, central nervous system diseases, pulmonary arterial hypertension.

15. A method of treating a tumor, a mitochondrial-associated disorder, a central nervous system disease, or pulmonary arterial hypertension, comprising:

15. The sirolimus-albumin composition of any one of claims 7-12 for use in the treatment of a patient or subject in need of treatment.

16. The composition of claim 13, the use of claim 14 or the method of claim 15, wherein the tumor is selected from the group consisting of liver cancer, lung cancer, pancreatic cancer, kidney cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, head and neck cancer, bladder cancer, malignant perivascular epithelioid cell tumor (PEComa), mantle cell lymphoma, multiple myeloma, lymphangioleiomyomatosis, Hodgkin's lymphoma.

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