Irinotecan hydrochloride liposome, and preparation process and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a liposome of irinotecan hydrochloride, a preparation process thereof and application. The preparation process comprises the following steps: (1) adjusting the concentration of sucrose octasulfate triethylamine and total lipids in an inner water phase, mixing the oil phase and the water phase according to certain flow rates and proportions through a microfluidic device to prepare blank liposomes; (2) using a HEPES buffer solution to remove free sucrose octasulfate triethylamine and organic solvents outside the blank liposomes, and concentrating; (3) using an active drug loading method to prepare drug-loaded liposomes. The scheme can greatly improve the encapsulation efficiency of the irinotecan hydrochloride liposome and accurately control the liposome particle size, and the finally prepared liposome particle size can be 90 nm-115 nm, the encapsulation efficiency reaches 95%, and the liposome has more excellent stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a liposome of irinotecan hydrochloride, a preparation process thereof and application thereof. BACKGROUND

[0002] Irinotecan (CPT 11) belongs to a semi-synthetic derivative of camptothecin and is a topoisomerase I inhibitor. Its mechanism of action is that irinotecan and its active metabolite SN-38 bind to a topoisomerase I-DNA complex to exert cytotoxic effects by causing DNA strand breaks. It is widely used in the clinical treatment of metastatic colorectal cancer, small cell lung cancer as a first / second line treatment, and pancreatic cancer as a first / second line treatment.

[0003] Currently marketed irinotecan products include irinotecan hydrochloride injection, lyophilized powder injection and liposome injection. Both irinotecan hydrochloride injection and lyophilized powder injection have relatively large toxic side effects. The reason is that the lactone ring structure of irinotecan is prone to hydrolysis. Under physiological conditions (pH 7.4), the equilibrium rapidly shifts to the carboxylate form, and the carboxylate form of irinotecan has low pharmacological activity and stronger toxicity, which ultimately leads to the discontinuation of chemotherapy for many patients.

[0004] Liposomes are a drug carrier that has been widely studied in recent years. The main feature is that it can protect the encapsulated drug and increase the stability of the drug. For anti-tumor liposomes, controlling the particle size of the liposomes at about 100 nm can effectively prolong the retention time of the liposomes in the tumor tissue, thereby better exerting the anti-tumor effect.

[0005] There are many international patents on irinotecan hydrochloride liposomes, but in terms of reducing and controlling particle size, most of them use a liposome extrusion instrument, such as CN102271659B and CN109260155B. The obtained liposome suspension is successively extruded through polycarbonate membranes with different pore sizes, and repeated multiple times to obtain the desired particle size of the liposomes. This process is prone to clogging the liposome extrusion instrument, which often needs to be repaired and cleaned. The polycarbonate membrane has a large consumption and is expensive. Other techniques for reducing the particle size of liposomes, such as ultrasonic fragmentation, are difficult to control in terms of power and time, and are only suitable for small volumes of liposomes to reduce particle size, which is difficult to scale up. High-pressure homogenizers are also prone to pipe blockage when reducing the particle size of liposomes, and the equipment is more complex and expensive, making it difficult to clean in the later stage. Shearing technology requires excellent control of energy, and improper control can easily cause damage to the liposomes, making it more suitable for the preparation of passive drug-loaded liposomes and multivesicular liposomes. In addition, the homogenization extrusion process may lead to instability of the liposomes, forming lipid fragments, thereby affecting the stability. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a liposome of irinotecan hydrochloride, a preparation process and application thereof. The purpose of the present application is to find a method which is convenient to operate, not easy to block the instrument pipeline, and can reduce the particle size of the liposome, and can batch produce the irinotecan hydrochloride liposome with a particle size of about 100 nm, a narrow particle size distribution, a high encapsulation efficiency, and a long-term stable storage. More desirably, the irinotecan hydrochloride liposome with an encapsulation efficiency of 95%, a particle size range of 90-115 nm, a PDI of less than 0.2, and a stable storage of at least 6 months can be batch produced. The high encapsulation efficiency can effectively protect the lactone ring structure of the irinotecan hydrochloride liposome, and the good particle size control can reduce the in vivo clearance of the irinotecan hydrochloride liposome, more penetrate into the tumor tissue, and improve the treatment effect.

[0007] The first aspect of the present application is to provide a liposome of irinotecan hydrochloride and a preparation process thereof, comprising the following steps:

[0008] Step S1: preparing an aqueous solution of sucrose octasulfate triethylamine as an aqueous phase, wherein the concentration of sucrose octasulfate triethylamine is 100-200 mM; dissolving liposome materials in an organic solvent to obtain an oil phase, wherein the total concentration of lipids is 22-34 mg / mL; and preparing blank liposomes by mixing the aqueous and oil phases through a microfluidic device;

[0009] Step S2: removing the free sucrose octasulfate triethylamine and the organic solvent outside the blank liposomes;

[0010] Step S3: dissolving irinotecan hydrochloride trihydrate in HEPES buffer, adding the blank liposomes according to the drug-lipid volume ratio of 1:1-1:3, carrying out active drug loading, and incubating at 50-70 DEG C to obtain the irinotecan hydrochloride liposome.

[0011] As a further optimization scheme of the preparation process of the irinotecan hydrochloride liposome, in step S1, the concentration of sucrose octasulfate triethylamine in the aqueous phase is 140-150 mM.

[0012] As a further optimization scheme of the preparation process of the irinotecan hydrochloride liposome, in step S1, the liposome materials are dissolved in ethanol to obtain the oil phase; the liposome materials include distearoylphosphatidylcholine (DSPC), cholesterol hemisuccinate hydrogenated vegetable oil ester (CHO-HP), and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine sodium salt (DSPE-mPEG2000); wherein the concentration of DSPC is 25.54 mg / mL, the concentration of CHO-HP is 8.33 mg / mL, and the concentration of DSPE-mPEG2000 is 0.45 mg / mL.

[0013] As a further optimization scheme of the preparation process of irinotecan hydrochloride liposomes, in step S1, the water-oil two phases are mixed by a microfluidic device, and the flow ratio of the oil-water two phases is controlled to be 1:(5±0.3).

[0014] As a further optimization scheme of the preparation process of irinotecan hydrochloride liposomes, in step S1, the oil-water two phases are mixed at 50-70℃, and after mixing, incubation is performed for 5-60 min. More preferably, the mixing is performed at 60-65℃, and after mixing, incubation is performed for 5-30 min.

[0015] As a further optimization scheme of the preparation process of irinotecan hydrochloride liposomes, in step S2, a HEPES buffer with a pH of 6.0-8.0 is used as the displacement medium, the free sucrose octasulfate triethylamine and organic solvent outside the liposomes are removed by a tangential flow filtration membrane device, and the blank liposomes are concentrated.

[0016] As a further optimization scheme of the preparation process of irinotecan hydrochloride liposomes, in step S2, the tangential flow filtration membrane device has a molecular weight cut-off of 100 kd.

[0017] As a further optimization scheme of the preparation process of irinotecan hydrochloride liposomes, in step S3, the concentration of irinotecan hydrochloride trihydrate is 2.0-30.0 mg / mL.

[0018] The second aspect of the present application provides irinotecan hydrochloride liposomes prepared according to the preparation process of the irinotecan hydrochloride liposomes described above, the prepared irinotecan hydrochloride liposomes have a particle size of 90-115 nm, a dispersion index of <0.2, and an encapsulation efficiency of ≥95%.

[0019] The third aspect of the present application provides the use of the preparation process of the irinotecan hydrochloride liposomes described above in the preparation of irinotecan hydrochloride liposome injections.

[0020] Advantages

[0021] The preparation method discovered by the present application can batch produce irinotecan hydrochloride liposomes with a particle size range of 90-115 nm, a PDI of <0.2, an encapsulation efficiency of 95%, and stable storage for at least 6 months. The high encapsulation efficiency can effectively protect the lactone ring structure of irinotecan hydrochloride liposomes, and good particle size control can reduce the in vivo clearance of irinotecan hydrochloride liposomes, more permeate into tumor tissues, and improve the therapeutic effect. The high encapsulation efficiency fully protects the lactone ring structure of the drug, avoids premature degradation; precise control of the particle size distribution of the liposomes reduces the risk of rapid clearance in vivo, allowing more drug to penetrate into tumor tissues to improve efficacy; excellent stability ensures consistent quality and efficacy of the product throughout the shelf life, facilitates transportation and storage, and facilitates clinical use.

[0022] The preparation process provided by the present application is not only convenient to operate, but also effectively avoids the problems of blockage, complex equipment and stability in traditional methods such as extrusion, ultrasonic and high-pressure homogenization, and can stably produce irinotecan hydrochloride liposomes on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 TEM image of irinotecan hydrochloride liposomes of Example 3.

[0024] Figure 2 In-vitro release profile of irinotecan hydrochloride liposomes of Example 3. DETAILED DESCRIPTION

[0025] The present application will be further illustrated by specific examples below, which are exemplary and intended to illustrate the problems and explain the present application, and are not a limitation.

[0026] Example 1

[0027] DSPC 61.29 mg, CHO-HP 19.98 mg and DSPE-mPEG2000 1.08 mg were weighed respectively, added with 2.4 mL of anhydrous ethanol, heated to 65°C to dissolve, as the oil phase, 140 mM sucrose octasulfate triethylamine 12 mL was preheated at 65°C, as the water phase. The reaction pipeline and microchannel reactor were placed in a 60°C environment for incubation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 50 mL / min:10 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The membrane was filtered to remove free sucrose octasulfate triethylamine outside the liposomes, and the liposomes were concentrated to 1 / 2 of the original volume to obtain irinotecan hydrochloride blank liposomes. Irinotecan hydrochloride trihydrate 24.51 mg was weighed and dissolved in 1.9 mL of HEPES buffer, and the drug-lipid ratio was 1:2 (v / v) to slowly drop into the blank liposomes, and incubated in a 55°C constant temperature water bath for 30 min to obtain irinotecan hydrochloride liposomes, which were stored in a 4°C refrigerator.

[0028] Example 2

[0029] DSPC 61.29 mg, CHO-HP 19.98 mg and DSPE-mPEG2000 1.08 mg were weighed respectively, 2.4 mL of anhydrous ethanol was added, and the mixture was dissolved by heating to 65 °C as an oil phase. 150 mM sucrose octasulfate triethylamine 12 mL was preheated at 65 °C as an aqueous phase. The reaction pipeline and microchannel reactor were incubated at 60 °C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 50 mL / min: 10 mL / min, and incubated for 5 min. The organic solvent and free sucrose octasulfate triethylamine outside the liposome were removed by membrane filtration, and the liposome was concentrated to 1 / 2 of the original volume to obtain blank irinotecan hydrochloride liposomes. 25.8 mg of irinotecan hydrochloride trihydrate was dissolved in 2 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:2 (v / v). The mixture was incubated in a 55 °C constant temperature water bath for 30 min to obtain irinotecan hydrochloride liposomes, which were stored in a refrigerator at 4 °C.

[0030] Example 3

[0031] DSPC 61.29 mg, CHO-HP 19.98 mg and DSPE-mPEG2000 1.08 mg were weighed respectively, 2.4 mL of anhydrous ethanol was added, and the mixture was dissolved by heating to 65 °C as an oil phase. 150 mM sucrose octasulfate triethylamine 12 mL was preheated at 65 °C as an aqueous phase. The reaction pipeline and microchannel reactor were incubated at 60 °C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 40 mL / min: 8 mL / min, and incubated for 5 min. The organic solvent and free sucrose octasulfate triethylamine outside the liposome were removed by membrane filtration, and the liposome was concentrated to 1 / 2 of the original volume to obtain blank irinotecan hydrochloride liposomes. 25.8 mg of irinotecan hydrochloride trihydrate was dissolved in 2 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:2 (v / v). The mixture was incubated in a 55 °C constant temperature water bath for 30 min to obtain irinotecan hydrochloride liposomes, which were stored in a refrigerator at 4 °C.

[0032] Comparative Example 1

[0033] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, molar ratio was 3:2:0.015, dissolved in 4 mL anhydrous ethanol, and the oil phase was slowly added into 4 mL 99.26 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and the resulting blank irinotecan hydrochloride liposomes were incubated in a 65°C constant temperature water bath for 20 min. The resulting lipid suspension was reduced in size using an ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by using a tangential flow filtration membrane with a 100KD cut-off, and 20% sucrose was used as the osmotic agent, added according to the volume ratio of 1:1, and the resulting blank liposomes were obtained. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise into the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated in a 60°C constant temperature water bath for 20 min. The resulting liposomes were stored in a 4°C refrigerator.

[0034] Comparative Example 2

[0035] DSPC (90.8 mg), CHOL (29.60 mg) and MPEG-2000-DSPE (1.60 mg) were weighed respectively, molar ratio was 3:2:0.015, dissolved in 1 mL anhydrous ethanol, and the oil phase was slowly added into 5 mL 99.26 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 5 mL with ultrapure water, and the resulting blank irinotecan hydrochloride liposomes were incubated in a 65°C constant temperature water bath for 20 min. The resulting lipid suspension was reduced in size using an ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by using a tangential flow filtration membrane with a 100KD cut-off, and 20% sucrose was used as the osmotic agent, added according to the volume ratio of 1:1, and the resulting blank liposomes were obtained. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise into the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated in a 55°C constant temperature water bath for 20 min. The resulting liposomes were stored in a 4°C refrigerator.

[0036] Comparative Example 3

[0037] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, the molar ratio was 3:2:0.015, dissolved in 4 mL of anhydrous ethanol, and the oil phase was slowly added to 4 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and incubated at 65°C for 20 min to obtain the irinotecan hydrochloride blank liposome. The obtained lipid suspension was reduced in size by ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by 100KD tangential flow filtration membrane, 20% sucrose was used as the osmotic agent, and the blank liposome was obtained after adding 1:1 by volume. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposome at a drug-lipid ratio of 1:3 (v / v), and incubated at 60°C for 20 min. The final liposome was stored in a refrigerator at 4°C.

[0038] Comparative Example 4

[0039] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, the molar ratio was 3:2:0.015, dissolved in 4 mL of anhydrous ethanol, and the oil phase was slowly added to 4 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and incubated at 65°C for 5 min to obtain the irinotecan hydrochloride blank liposome. The obtained lipid suspension was reduced in size by ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by tangential flow filtration membrane, 20% sucrose was used as the osmotic agent, and the blank liposome was obtained after adding 1:1 by volume. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposome at a drug-lipid ratio of 1:3 (v / v), and incubated at 55°C for 20 min. The final liposome was stored in a refrigerator at 4°C.

[0040] Comparative Example 5

[0041] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, the molar ratio was 3:2:0.015, dissolved in 4 mL of anhydrous ethanol, and the oil phase was slowly added to 4 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and incubated at 65°C for 60 min to obtain irinotecan hydrochloride blank liposomes. The obtained lipid suspension was reduced in size by ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by tangential flow filtration membrane, 20% sucrose was used as the osmotic agent, and the blank liposomes were obtained after adding 1:1 by volume. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated at 55°C for 20 min. The final liposomes were stored in a refrigerator at 4°C.

[0042] Comparative Example 6

[0043] DSPC (61.29 mg), CHOL (19.98 mg) and MPEG-2000-DSPE (1.08 mg) were weighed respectively, the molar ratio was 3:2:0.015, dissolved in 3 mL of anhydrous ethanol, and the oil phase was slowly added to 3 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 3 mL with ultrapure water, and incubated at 65°C for 20 min to obtain irinotecan hydrochloride blank liposomes. The obtained lipid suspension was reduced in size by ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by tangential flow filtration membrane, 20% sucrose was used as the osmotic agent, and the blank liposomes were obtained after adding 1:1 by volume. 25.8 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 2 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:2 (v / v), and incubated at 55°C for 20 min. The final liposomes were stored in a refrigerator at 4°C.

[0044] Comparative Example 7

[0045] DSPC (51.08 mg), CHOL (16.65 mg) and MPEG-2000-DSPE (0.90 mg) were weighed respectively, the molar ratio was 3:2:0.015, and they were dissolved in 3 mL of anhydrous ethanol. The oil phase was slowly added to 3 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 3 mL with ultrapure water, and incubated at 65°C for 20 min to obtain blank irinotecan hydrochloride liposomes. The obtained lipid suspension was reduced in size by an ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by tangential flow filtration membrane, and 20% sucrose was used as the osmotic agent, and the volume was added to 1:1 to obtain blank liposomes. 21.5 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:4 (v / v), and incubated at 55°C for 20 min. The final liposomes were stored in a refrigerator at 4°C.

[0046] Comparative Example 8

[0047] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, the molar ratio was 3:2:0.015, and they were dissolved in 4 mL of anhydrous ethanol. The oil phase was slowly added to 4 mL of 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and incubated at 55°C for 20 min to obtain blank irinotecan hydrochloride liposomes. The obtained lipid suspension was reduced in size by an ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the external aqueous phase sucrose octasulfate triethylamine was removed by tangential flow filtration membrane, and 20% sucrose was used as the osmotic agent, and the volume was added to 1:1 to obtain blank liposomes. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated at 55°C for 20 min. The final liposomes were stored in a refrigerator at 4°C.

[0048] Comparative Example 9

[0049] DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed separately, molar ratio was 3:2:0.015, dissolved in 4 mL anhydrous ethanol, the oil phase was slowly added to 4 mL 81.25 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing ethanol by evaporation under reduced pressure, the volume was made up to 4 mL with ultrapure water, and incubated at 75°C for 20 min to obtain irinotecan hydrochloride blank liposomes. The resulting lipid suspension was reduced in size using an ultrasonic cell crusher, power: 30%, ultrasonic for 30 min; power: 60%, ultrasonic for 25 min. Then the sucrose octasulfate triethylamine in the external aqueous phase was removed by tangential flow filtration membrane, 20% sucrose was used as the osmotic agent, and the blank liposomes were obtained after adding 1:1 (v / v) by volume. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated at 55°C for 20 min. The final liposomes were stored in a refrigerator at 4°C.

[0050] Comparative Example 10

[0051] DSPC 72.64 mg, CHO-HP 23.68 mg and DSPE-mPEG2000 1.28 mg were weighed separately, added to 4 mL anhydrous ethanol, and dissolved by heating to 65°C, as the oil phase. 400 mM sucrose octasulfate triethylamine 4 mL was preheated at 65°C as the aqueous phase. The reaction pipeline and microchannel reactor were kept at 60°C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 5 mL / min:5 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The free sucrose octasulfate triethylamine outside the liposomes was removed by membrane filtration to obtain irinotecan hydrochloride blank liposomes. 17.2 mg of irinotecan hydrochloride trihydrate was weighed and dissolved in 1 mL of HEPES buffer, and then slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v), and incubated at 55°C for 20 min. The irinotecan hydrochloride liposomes were obtained and stored in a refrigerator at 4°C.

[0052] Comparative Example 11

[0053] DSPC 72.64 mg, CHO-HP 23.68 mg and DSPE-mPEG2000 1.28 mg were weighed respectively, added with 4 mL of anhydrous ethanol, and dissolved by heating to 65 °C as the oil phase. 4 mL of 400 mM sucrose octasulfate triethylamine preheated at 65 °C was used as the water phase. The reaction pipeline and microchannel reactor were kept at 60 °C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 20 mL / min: 20 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The free sucrose octasulfate triethylamine outside the liposomes was removed by membrane filtration to obtain blank irinotecan hydrochloride liposomes. 17.2 mg of irinotecan hydrochloride trihydrate was dissolved in 1 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v). The mixture was incubated in a 55 °C constant temperature water bath for 20 min to obtain irinotecan hydrochloride liposomes, which were stored in a refrigerator at 4 °C.

[0054] Comparative Example 12

[0055] DSPC 54.48 mg, CHO-HP 17.76 mg and DSPE-mPEG2000 0.96 mg were weighed respectively, added with 2 mL of anhydrous ethanol, and dissolved by heating to 65 °C as the oil phase. 6 mL of 400 mM sucrose octasulfate triethylamine preheated at 65 °C was used as the water phase. The reaction pipeline and microchannel reactor were kept at 60 °C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 21 mL / min: 7 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The free sucrose octasulfate triethylamine outside the liposomes was removed by membrane filtration to obtain blank irinotecan hydrochloride liposomes. 17.2 mg of irinotecan hydrochloride trihydrate was dissolved in 1 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:3 (v / v). The mixture was incubated in a 55 °C constant temperature water bath for 20 min to obtain irinotecan hydrochloride liposomes, which were stored in a refrigerator at 4 °C.

[0056] Comparative Example 13

[0057] Take 54.48 mg of DSPC, 17.76 mg of CHO-HP and 0.96 mg of DSPE-mPEG20000, respectively, add 2 mL of anhydrous ethanol, heat to 65°C to dissolve, as the oil phase, 6 mL of 400 mM sucrose octasulfate triethylamine preheated at 65°C as the water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, and the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 42 mL / min: 14 mL / min, and the organic solvent is removed by evaporation under reduced pressure to obtain a liposome suspension, which is incubated for 20 min. The membrane bag is filtered to remove the free sucrose octasulfate triethylamine outside the liposomes to obtain irinotecan hydrochloride blank liposomes. Take 17.2 mg of irinotecan hydrochloride trihydrate, dissolve in 1 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:3 (v / v), incubate in a 55°C constant temperature water bath for 20 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0058] Comparative Example 14

[0059] Take 54.48 mg of DSPC, 17.76 mg of CHO-HP and 0.96 mg of DSPE-mPEG20000, respectively, add 2 mL of anhydrous ethanol, heat to 65°C to dissolve, as the oil phase, 6 mL of 400 mM sucrose octasulfate triethylamine preheated at 65°C as the water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, and the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 42 mL / min: 14 mL / min, and the organic solvent is removed by evaporation under reduced pressure to obtain a liposome suspension, which is incubated for 20 min. The membrane bag is filtered to remove the free sucrose octasulfate triethylamine outside the liposomes to obtain irinotecan hydrochloride blank liposomes. Take 17.2 mg of irinotecan hydrochloride trihydrate, dissolve in 1 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:3 (v / v), incubate in a 55°C constant temperature water bath for 20 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0060] Comparative Example 15

[0061] Take DSPC 45.97 mg, CHO-HP 15.99 mg and DSPE-mPEG20000 0.81 mg respectively, add 3 mL of anhydrous ethanol, heat to 65°C to dissolve, as oil phase, 225 mM sucrose octasulfate triethylamine 9 mL preheated at 65°C, as water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 66 mL / min:22 mL / min, the organic solvent is removed by evaporation under reduced pressure, and a liposome suspension is obtained, which is incubated for 20 min. The membrane bag filter is used to remove the free sucrose octasulfate triethylamine outside the liposomes, and the liposomes are concentrated to 1 / 2 of the original volume to obtain irinotecan hydrochloride blank liposomes. Take 24.51 mg of irinotecan hydrochloride trihydrate, dissolve in 1.9 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:2 (v / v), incubate in a 55°C constant temperature water bath for 30 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0062] Comparative Example 16

[0063] Take DSPC 45.97 mg, CHO-HP 15.99 mg and DSPE-mPEG20000 0.81 mg respectively, add 3 mL of anhydrous ethanol, heat to 65°C to dissolve, as oil phase, 165 mM sucrose octasulfate triethylamine 9 mL preheated at 65°C, as water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 66 mL / min:22 mL / min, the organic solvent is removed by evaporation under reduced pressure, and a liposome suspension is obtained, which is incubated for 20 min. The membrane bag filter is used to remove the free sucrose octasulfate triethylamine outside the liposomes, and the liposomes are concentrated to 1 / 2 of the original volume to obtain irinotecan hydrochloride blank liposomes. Take 24.51 mg of irinotecan hydrochloride trihydrate, dissolve in 1.9 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:2 (v / v), incubate in a 55°C constant temperature water bath for 30 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0064] Comparative Example 17

[0065] Take DSPC 45.97 mg, CHO-HP 15.99 mg and DSPE-mPEG20000 0.81 mg respectively, add 3 mL of anhydrous ethanol, heat to 65°C to dissolve, as oil phase, 94 mM sucrose octasulfate triethylamine 9 mL preheated at 65°C, as water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 66 mL / min:22 mL / min, the organic solvent is removed by evaporation under reduced pressure, and a liposome suspension is obtained, which is incubated for 20 min. Membrane bag filtration is used to remove the free sucrose octasulfate triethylamine outside the liposomes, and the liposomes are concentrated to 1 / 2 of the original volume, to obtain blank irinotecan hydrochloride liposomes. Take 24.51 mg of irinotecan hydrochloride trihydrate, dissolve in 1.9 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:2 (v / v), incubate in a 55°C constant temperature water bath for 30 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0066] Comparative Example 18

[0067] Take DSPC 45.97 mg, CHO-HP 15.99 mg and DSPE-mPEG20000 0.81 mg respectively, add 3 mL of anhydrous ethanol, heat to 65°C to dissolve, as oil phase, 94 mM sucrose octasulfate triethylamine 9 mL preheated at 65°C, as water phase. The reaction pipeline and microchannel reactor are placed in a 60°C environment for incubation, the water and oil phases are mixed through the microchannel reactor at a flow rate ratio of 66 mL / min:22 mL / min, the organic solvent is removed by evaporation under reduced pressure, and a liposome suspension is obtained, which is incubated for 20 min. Membrane bag filtration is used to remove the free sucrose octasulfate triethylamine outside the liposomes, and the liposomes are concentrated to 1 / 2 of the original volume, to obtain blank irinotecan hydrochloride liposomes. Take 24.51 mg of irinotecan hydrochloride trihydrate, dissolve in 1.9 mL of HEPES buffer, and slowly drop into the blank liposomes at a drug-lipid ratio of 1:2 (v / v), incubate in a 55°C constant temperature water bath for 30 min, and obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0068] Comparative Example 19

[0069] DSPC 40.86 mg, CHO-HP 13.32 mg and DSPE-mPEG20000 0.72 mg were weighed respectively, 2.4 mL of anhydrous ethanol was added, and the mixture was dissolved by heating to 65°C, as the oil phase, 140 mM sucrose octasulfate triethylamine 12 mL was preheated at 65°C as the water phase. The reaction pipeline and microchannel reactor were kept at 60°C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 50 mL / min: 10 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 3 of the original volume to obtain irinotecan hydrochloride blank liposomes. 24.51 mg of irinotecan hydrochloride trihydrate was dissolved in 1.9 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:2 (v / v). The mixture was incubated in a 55°C constant temperature water bath for 30 min to obtain irinotecan hydrochloride liposomes, which were stored in a 4°C refrigerator.

[0070] Comparative Example 20

[0071] DSPC 54.48 mg, CHO-HP 17.76 mg and DSPE-mPEG20000 0.96 mg were weighed respectively, 2.4 mL of anhydrous ethanol was added, and the mixture was dissolved by heating to 65°C, as the oil phase, 140 mM sucrose octasulfate triethylamine 12 mL was preheated at 65°C as the water phase. The reaction pipeline and microchannel reactor were kept at 60°C. The water and oil phases were mixed through the microchannel reactor at a flow rate ratio of 50 mL / min: 10 mL / min. The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20 min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 of the original volume to obtain irinotecan hydrochloride blank liposomes. 24.51 mg of irinotecan hydrochloride trihydrate was dissolved in 1.9 mL of HEPES buffer, and was slowly added dropwise to the blank liposomes at a drug-lipid ratio of 1:2 (v / v). The mixture was incubated in a 55°C constant temperature water bath for 30 min to obtain irinotecan hydrochloride liposomes, which were stored in a 4°C refrigerator.

[0072] Test Example 1: Test of liposome particle size

[0073] An appropriate amount of liposomes prepared in Examples 1-3 and Comparative Examples 1-20 was diluted with distilled water, and the particle size and PDI were determined using a laser particle size analyzer (ZLS). The above experiment was determined three times, and the average value was taken, and the results are shown in Table 1.

[0074] Table 1 Liposome particle size and PDI of different examples and comparative examples

[0075]

[0076]

[0077] Examples 1 to 3 all used microfluidic equipment to prepare irinotecan hydrochloride liposomes, and the oil-water two-phase ratio was controlled to be 1:5±0.3, wherein the DSPC concentration was 25.54 mg / mL, the CHO-HP concentration was 8.33 mg / mL, the DSPE-mPEG2000 concentration was 0.45 mg / mL; and the sucrose octasulfate triethylamine concentration was adjusted to 140-150 mM.

[0078] The results show that the irinotecan hydrochloride liposomes prepared in Examples 1 to 3 have particle sizes controlled between 90-115 nm, and PDI (polydispersity index) is less than 0.2, indicating that the particle size of the prepared irinotecan hydrochloride liposomes is well controlled at about 100 nm, has excellent monodispersity and uniform particle size distribution, and helps to prolong the residence time of irinotecan hydrochloride liposomes in tumor tissue, thereby better exerting the anti-tumor effect. The TEM (transmission electron microscope) image of the irinotecan hydrochloride liposomes prepared in Example 3 is shown in FIG. 1, which shows that the liposome morphology is regular, the size is uniform, and the diameter is about 100 nm. Figure 1

[0079] Comparative Examples 1 to 9 used a dropwise addition method to add the oil phase to the water phase, and used ultrasonic crushing to reduce the liposome particle size. It was found that by adjusting the amount of DSPC, the amount of CHOL, the amount of MPEG-2000-DSPE, the amount of anhydrous ethanol, the concentration of sucrose octasulfate triethylamine, the amount of sucrose octasulfate triethylamine, the incubation time, the incubation temperature, the amount of irinotecan hydrochloride, and the drug-lipid ratio, only Comparative Example 3 achieved good particle size and distribution, and other samples could not prepare irinotecan hydrochloride liposomes with particle sizes between 90-115 nm and PDI less than 0.2.

[0080] Comparative Examples 10 to 20 used a method similar to Examples 1 to 3, and also used the same microfluidic equipment to prepare irinotecan hydrochloride liposomes. The oil-water ratio and the concentration of the initial lipids were changed, but it was found that no matter how the scheme was adjusted, irinotecan hydrochloride liposomes with particle sizes between 90-115 nm and PDI less than 0.2 could not be prepared.

[0081] Test Example 2: Test of liposome encapsulation efficiency

[0082] ​Take 1 ml of the irinotecan hydrochloride liposomes prepared in Example 1-3, Comparative Example 1-20 in a dialysis bag, and place 600 ml of water outside. After dialysis for 6 h under stirring, take the dialysate and determine the content of irinotecan hydrochloride liposomes by HPLC to obtain Wout. Take another 1 ml of irinotecan hydrochloride liposomes in a 100 ml volumetric flask, and add methanol to constant volume. Sonicate to break the emulsion, and analyze by HPLC to obtain Wtotal. The encapsulation efficiency formula is EE = (Wtotal - Wout) / Wtotal x 100%.

[0083] Table 2 Liposome particle size of different examples and comparative examples

[0084]

[0085]

[0086] Examples 1 to 3 all use microfluidic equipment to prepare irinotecan hydrochloride liposomes, and the oil-water two-phase ratio is controlled to be 1:5±0.3, wherein the DSPC concentration is 25.54 mg / mL, the CHOL concentration is 8.33 mg / mL, and the DSPE-mPEG2000 concentration is 0.45 mg / mL. The prepared irinotecan hydrochloride liposomes can all achieve a high encapsulation efficiency, and the encapsulation efficiency can reach 95%.

[0087] Especially, using the formulation of Example 3, after mixing the oil-water two phases through the microfluidic device, the incubation of the blank liposomes only needs 5 min, and then the ethanol and free internal water phase are removed by membrane packaging, so that irinotecan hydrochloride liposomes with an encapsulation efficiency of more than 95% can be obtained. Therefore, Example 3 has a more simple process in actual implementation, and has a significant advantage in process amplification.

[0088] Comparative Examples 1 to 9 use the ethanol injection method combined with ultrasonic crushing to reduce the particle size to prepare irinotecan hydrochloride liposomes. It is found that by adjusting the amount of DSPC, the amount of CHOL, the amount of MPEG-2000-DSPE, the amount of anhydrous ethanol, the concentration of triethylamine sucrose octasulfate, the amount of triethylamine sucrose octasulfate, the incubation time, the incubation temperature, the amount of irinotecan hydrochloride, and the drug-lipid ratio, the encapsulation efficiency of the liposomes prepared in other cases is not high, and generally difficult to reach 90%. Comparative Examples 10 to 20 use a method similar to Examples 1 to 3, but due to the change of the prescription, the encapsulation efficiency is generally not high.

[0089] Test Example 3: Test the in vitro release rate of irinotecan hydrochloride liposomes

[0090] Take 1 mL of the liposome of Example 3, put it into a dialysis bag of 8000-14000 Da, seal it, pour it into 100 mL of release medium, which is a PBS solution containing 10 mM ammonium sulfate at pH 6.5, and put it into a 37°C water bath constant temperature shaking incubator. At 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, take out part of the release medium and replace it with fresh release medium. The released medium is analyzed by HPLC to calculate the cumulative release percentage.

[0091] As shown in Figure 2 , the irinotecan hydrochloride liposome is slowly released in the release medium, with about 50% released at 24 h. Sucrose octasulfate and irinotecan hydrochloride can form a precipitate in the inner aqueous phase, improving the retention of irinotecan hydrochloride in the liposome and significantly slowing down the drug release rate, with excellent sustained release effect.

[0092] Test Example 4: Test the stability of irinotecan hydrochloride liposome

[0093] Take the liposome of Example and the liposome of Comparative Example 3 and place them in a 2-8°C environment, observe the physical state of the sample regularly, and investigate the product stability. The results are shown in Table 3.

[0094] Table 3 Product stability record

[0095]

[0096] The results show that the irinotecan hydrochloride liposome preparation of Example maintains good stability throughout the 6-month observation period, always maintaining a light yellow translucent suspension state, without obvious physical changes or precipitation. This indicates that the liposome prepared by microfluidic device and strictly controlling the oil-water two-phase ratio, initial lipid concentration, and other parameters has excellent long-term stability. In contrast, the liposome of Comparative Example 3, although initially also presents a light yellow translucent suspension, has a very small amount of precipitation after 1 month of storage, and the precipitation phenomenon becomes more obvious as the time extends to 3 months and 6 months. This shows that although Comparative Example 3 achieves good particle size and distribution, there are still deficiencies in the overall formulation and / or preparation method, which fail to achieve the long-term stability of irinotecan hydrochloride liposome.

[0097] The above embodiments are exemplary and are intended to illustrate the technical concept and characteristics of the present application, so that those skilled in the art can understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A process for the preparation of liposomal irinotecan hydrochloride characterized in that: Comprising the following steps: Step S1: preparing sucrose octasulfate triethylamine aqueous solution as the water phase, wherein the concentration of sucrose octasulfate triethylamine is 100-200 mM; dissolving liposome material in an organic solvent to obtain the oil phase, wherein the total concentration of lipids is 22-34 mg / mL; and mixing the water and oil phases by a microfluidic device to obtain blank liposomes; Step S2: removing sucrose octasulfate triethylamine and organic solvent outside the blank liposomes; Step S3: dissolving irinotecan hydrochloride trihydrate in HEPES buffer, adding the blank liposomes according to the drug-lipid volume ratio of 1:1-1:3, performing active drug loading, and incubating at 50-70°C to obtain irinotecan hydrochloride liposomes; In step S1, the concentration of sucrose octasulfate triethylamine in the water phase is 140-150 mM; In step S1, the water and oil phases are mixed by a microfluidic device, and the flow ratio of the oil and water phases is controlled to be 1:(5±0.3); In step S1, the liposome material is dissolved in ethanol to obtain the oil phase; the liposome material comprises distearoylphosphatidylcholine (DSPC), cholesterol hemisuccinate hydrogenated vegetable oil ester (CHO-HP), and N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine sodium salt (DSPE-mPEG2000); wherein the concentration of DSPC is 25.54 mg / mL, the concentration of CHO-HP is 8.33 mg / mL, and the concentration of DSPE-mPEG2000 is 0.45 mg / mL; In step S3, the concentration of irinotecan hydrochloride trihydrate is 2.0-30.0 mg / mL.

2. The process for the preparation of liposomal irinotecan hydrochloride as claimed in claim 1, wherein: In step S1, the mixing of the oil and water phases is performed at 50-70°C, and the mixture is incubated for 5-60 min after mixing.

3. The process for the preparation of liposomal irinotecan hydrochloride according to claim 1 or 2, characterized in that: In step S2, a HEPES buffer with a pH of 6.0-8.0 is used as the displacement medium, and a tangential flow filtration membrane device is used to remove sucrose octasulfate triethylamine and organic solvent outside the blank liposomes and concentrate the blank liposomes.

4. The process for the preparation of liposomal irinotecan hydrochloride as claimed in claim 3, wherein the process comprises: In step S2, the tangential flow filtration membrane device has a molecular weight cut-off of 100 kd.

5. A liposome of irinotecan hydrochloride, characterized by: The irinotecan hydrochloride liposomes are prepared according to the preparation process of the irinotecan hydrochloride liposomes according to any one of claims 1-4; the particle size of the irinotecan hydrochloride liposomes is 90-115 nm, the dispersity index is <0.2, and the encapsulation efficiency is ≥95%.

6. The irinotecan hydrochloride liposomes according to claim 5 for use in the preparation of irinotecan hydrochloride liposome injections.

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