Irinotecan hydrochloride liposome as well as preparation process and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an irinotecan hydrochloride liposome as well as a preparation process and application thereof. The preparation process comprises the following steps: (1) adjusting the concentration of sucrose octasulfate triethylamine and total lipid in an inner water phase, and mixing an oil phase and a water phase according to a certain flow velocity and proportion by virtue of microfluidic equipment, so as to prepare a blank liposome; (2) removing free sucrose octasulfate triethylamine and an organic solvent in the blank liposome by using an HEPES buffer solution, and concentrating; and (3) preparing the drug-loaded liposome by adopting an active drug loading method. According to the scheme, the encapsulation efficiency of the irinotecan hydrochloride lipidosome can be greatly improved, the particle size of the lipidosome can be accurately controlled, the particle size of the finally prepared lipidosome can range from 90 nm to 115 nm, the encapsulation efficiency reaches 95%, and meanwhile more excellent stability is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to irinotecan hydrochloride liposomes and a preparation process and application thereof. Background Art

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

[0003] Currently available irinotecan products include irinotecan hydrochloride injection, lyophilized powder injection and liposome injection. Both irinotecan hydrochloride injection and lyophilized powder injection have significant toxic side effects because the lactone ring structure of irinotecan is easily hydrolyzed. Under physiological conditions (pH 7.4), the equilibrium quickly shifts to the carboxylate type. The carboxylate type of irinotecan has low pharmacological activity and is more toxic, which ultimately makes chemotherapy impossible for many patients.

[0004] As a drug carrier that has been widely studied in recent years, liposomes are mainly characterized by their ability to protect encapsulated drugs and increase drug stability. For anti-tumor liposomes, controlling the liposome particle size to around 100nm can effectively prolong the retention time of liposomes in tumor tissues, thereby better exerting anti-tumor efficacy.

[0005] There are also many international patents on irinotecan hydrochloride liposomes, but in terms of reducing and controlling particle size, liposome extruders are mostly used, such as CN102271659B, CN109260155B, etc. The obtained liposome suspension is sequentially extruded through polycarbonate membranes of different pore sizes, and repeated multiple times to obtain the desired liposome particle size. This process is very easy to block the liposome extruder, and often requires maintenance and cleaning. The polycarbonate membrane consumes a lot of energy and is expensive. Other technologies that reduce the particle size of liposomes, such as ultrasonic crushing, are only suitable for small-volume liposomes to reduce the particle size because the power and time are difficult to control, and the process is difficult to scale up. High-pressure homogenizers are also prone to pipe blocking when reducing the particle size of liposomes, and the equipment is more complex and expensive, and it is difficult to clean up later. Shearing technology requires excellent control of energy, and improper control can easily cause damage to liposomes, which is more suitable for the preparation of passive drug-loaded liposomes and multivesicular liposomes. In addition, the homogenization extrusion process may cause the liposomes to be unstable and form lipid fragments, thereby affecting stability. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides an irinotecan hydrochloride liposome and a preparation process and application thereof. The purpose of the present invention is to find a method for reducing the particle size of liposomes that is easy to operate and not easy to clog instrument pipelines, and to be able to batch prepare irinotecan hydrochloride liposomes with a particle size of about 100nm, a narrow particle size distribution, a high encapsulation rate, and long-term stable storage. Ideally, irinotecan hydrochloride liposomes with an encapsulation rate of 95%, a particle size range of 90 to 115nm, PDI <0.2, and stable storage for at least 6 months can be prepared in batches. The high encapsulation rate can effectively protect the lactone ring structure of the irinotecan hydrochloride liposome, and good particle size control can reduce the in vivo clearance of the irinotecan hydrochloride liposome, penetrate more into tumor tissues, and improve the therapeutic effect.

[0007] The first aspect of the present invention is to provide an irinotecan hydrochloride liposome 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 a liposome material in an organic solvent as an oil phase, wherein the total lipid concentration is 22-34 mg / mL; mixing the water-oil phases by a microfluidic device to prepare blank liposomes;

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

[0010] Step S3: Dissolve irinotecan hydrochloride trihydrate in HEPES buffer, add it into blank liposomes at a drug-lipid volume ratio of 1:1 to 1:3 for active drug loading, and incubate at 50 to 70° C. to obtain irinotecan hydrochloride liposomes.

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

[0012] As a further optimization scheme for the preparation process of irinotecan hydrochloride liposomes, in step S1, the liposome material is dissolved in ethanol to obtain an oil phase; the liposome material includes distearoylphosphatidylcholine (DSPC), cholesterol hemisuccinate hydrogenated vegetable oil ester (CHO-HP), and N-(carbonyl monomethoxy polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-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 for 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 for the preparation process of irinotecan hydrochloride liposomes, in step S1, the oil-water two-phase is mixed at 50-70° C., and then incubated for 5-60 minutes. More preferably, the mixing is carried out at 60-65° C., and then incubated for 5-30 minutes.

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

[0016] As a further optimization scheme for the preparation process of irinotecan hydrochloride liposomes, in step S2, the molecular weight cutoff of the tangential flow filtration membrane package device is 100 kd.

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

[0018] The second aspect of the present invention is to provide an irinotecan hydrochloride liposome, which is prepared according to the above-mentioned preparation process of the irinotecan hydrochloride liposome. The prepared irinotecan hydrochloride liposome has a particle size of 90-115nm, a dispersibility index of <0.2, and an encapsulation rate of ≥95%.

[0019] The third aspect of the present invention is to provide application of the above-mentioned preparation process of irinotecan hydrochloride liposome in the preparation of irinotecan hydrochloride liposome injection.

[0020] Beneficial Effects

[0021] The preparation method discovered by the present invention can batch prepare irinotecan hydrochloride liposomes with a particle size range of 90 to 115 nm, PDI < 0.2, an encapsulation rate of 95%, and stable storage for at least 6 months. The high encapsulation rate can effectively protect the lactone ring structure of the irinotecan hydrochloride liposomes, and good particle size control can reduce the in vivo clearance of the irinotecan hydrochloride liposomes, allowing more drugs to penetrate into tumor tissues and improve the therapeutic effect. The high encapsulation rate fully protects the lactone ring structure of the drug to avoid premature degradation; the precise control of the particle size distribution of the liposomes reduces the risk of rapid clearance in the body, allowing more drugs to penetrate into tumor tissues to improve the efficacy; the excellent stability ensures that the product maintains consistent quality and efficacy throughout the entire validity period, is convenient for transportation and storage, and is convenient for clinical use.

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

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

[0024] Figure 2 This is the in vitro release diagram of irinotecan hydrochloride liposomes in Example 3. DETAILED DESCRIPTION

[0025] The present invention is further illustrated by specific examples below. These examples are exemplary and are intended to illustrate the problem and explain the present invention, but are not intended to be limiting.

[0026] Example 1

[0027] Weigh 61.29 mg of DSPC, 19.98 mg of CHO-HP, and 1.08 mg of DSPE-mPEG2000, respectively, add 2.4 mL of anhydrous ethanol, heat to 65 ° C to dissolve, as the oil phase, 12 mL of 140 mM sucrose octasulfate triethylamine preheated at 65 ° C, as the water phase. The reaction pipeline and microchannel reactor were placed at 60 ° C for insulation, 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 minutes. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposomes were concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0028] Example 2

[0029] DSPC 61.29 mg, CHO-HP 19.98 mg and DSPE-mPEG20001.08 mg were weighed, 2.4 mL of anhydrous ethanol was added, and the mixture was heated to 65 ° C to dissolve as the oil phase. 150 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 insulation, and 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 minutes. The organic solvent and the free sucrose octasulfate triethylamine outside the liposomes were removed by membrane filtration, and the liposomes were concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 25.8 mg of irinotecan hydrochloride trihydrate, dissolve it in 2 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0030] Example 3

[0031] DSPC 61.29 mg, CHO-HP 19.98 mg and DSPE-mPEG20001.08 mg were weighed, 2.4 mL of anhydrous ethanol was added, and the mixture was heated to 65 ° C to dissolve as the oil phase. 12 mL of 150 mM sucrose octasulfate triethylamine was preheated at 65 ° C as the water phase. The reaction pipeline and microchannel reactor were placed in a 60 ° C environment for insulation, and 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 minutes. The organic solvent and the free sucrose octasulfate triethylamine outside the liposomes were removed by membrane filtration, and the liposomes were concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 25.8 mg of irinotecan hydrochloride trihydrate, dissolve it in 2 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0032] Comparative Example 1

[0033] Due to the different batches supported by different processes, in order to maintain the oil phase composition similar to that of the embodiment, DSPC (72.64 mg), CHOL (23.68 mg) and MPEG-2000-DSPE (1.28 mg) were weighed respectively, with a molar ratio of 3:2:0.015, dissolved in 4 mL of anhydrous ethanol, and the oil phase was slowly added dropwise to 4 mL of 99.26 mM sucrose octasulfate triethylamine in a 65°C hot water bath. After removing the ethanol by reduced pressure evaporation, the volume was supplemented to 4 ml with ultrapure water, and incubated in a 65°C constant temperature water bath for 20 min to obtain irinotecan hydrochloride blank liposomes. The obtained lipid suspension was reduced in particle size by an ultrasonic cell crusher, power: 30%, ultrasonic 30 min; power: 60% ultrasonic 25 min. Then, a tangential flow filtration membrane package 100KD was used to remove the external aqueous phase sucrose octasulfate triethylamine, and 20% sucrose was used as an osmotic agent, and blank liposomes were obtained after adding it in a volume ratio of 1:1. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 ml of HEPES buffer, and slowly add it dropwise into the blank liposomes at a drug-lipid ratio of 1:3 (v / v). Incubate in a 60°C constant temperature water bath for 20 min. The final liposomes are 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, with a molar ratio of 3:2:0.015, dissolved in 1 mL of anhydrous ethanol, and the oil phase was slowly added dropwise to 5 ml of 99.26 mM sucrose octasulfate triethylamine in a 65 °C hot water bath. After removing the ethanol by evaporation under reduced pressure, the volume was supplemented with ultrapure water to 5 ml, and incubated in a 65 °C constant temperature water bath for 20 min to obtain irinotecan hydrochloride blank liposomes. The obtained lipid suspension was reduced in particle size by ultrasonic cell crusher, power: 30%, ultrasonic 30 min; power: 60% ultrasonic 25 min. Then, tangential flow filtration membrane package 100KD was used to remove the external aqueous phase sucrose octasulfate triethylamine, and 20% sucrose was used as an osmotic agent, and blank liposomes were obtained after adding it in a volume ratio of 1:1. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 ml of HEPES buffer, and slowly add it dropwise 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. The final liposomes are 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, with a molar ratio of 3:2:0.015, dissolved in 4 mL of anhydrous ethanol, and the oil phase was slowly added dropwise to 4 mL of 81.25 mM sucrose octasulfate triethylamine in a 65 °C hot water bath. After removing the ethanol by evaporation under reduced pressure, the volume was supplemented to 4 ml with ultrapure water, and incubated in a 65 °C constant temperature water bath for 20 min to obtain irinotecan hydrochloride blank liposomes. The obtained lipid suspension was reduced in particle size by ultrasonic cell crusher, power: 30%, ultrasonic 30 min; power: 60% ultrasonic 25 min. Then, tangential flow filtration membrane package 100KD was used to remove the external aqueous phase sucrose octasulfate triethylamine, and 20% sucrose was used as an osmotic agent, and blank liposomes were obtained after adding it in a volume ratio of 1:1. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 ml of HEPES buffer, and slowly add it dropwise into the blank liposomes at a drug-lipid ratio of 1:3 (v / v). Incubate in a 60°C constant temperature water bath for 20 min. The final liposomes are stored in a 4°C refrigerator.

[0038] Comparative Example 4

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

[0040] Comparative Example 5

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

[0042] Comparative Example 6

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

[0044] Comparative Example 7

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

[0046] Comparative Example 8

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

[0048] Comparative Example 9

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

[0050] Comparative Example 10

[0051] DSPC 72.64mg, CHO-HP 23.68mg and DSPE-mPEG20001.28mg were weighed respectively, 4mL of anhydrous ethanol was added, and the mixture was heated to 65℃ to dissolve as the oil phase. 4mL of 400mM sucrose octasulfate triethylamine was preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (5mL / min:5mL / min). The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration to obtain blank irinotecan hydrochloride liposomes. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are then stored in a 4°C refrigerator.

[0052] Comparative Example 11

[0053] Weigh 72.64 mg of DSPC, 23.68 mg of CHO-HP, and 1.28 mg of DSPE-mPEG2000, respectively, add 4 mL of anhydrous ethanol, heat to 65 ° C to dissolve, as the oil phase, 400 mM sucrose octasulfate triethylamine 4 mL preheated at 65 ° C, as the water phase. The reaction pipeline and microchannel reactor were placed at 60 ° C for insulation, and 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 minutes. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration to obtain irinotecan hydrochloride blank liposomes. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are then stored in a 4°C refrigerator.

[0054] Comparative Example 12

[0055] DSPC 54.48mg, CHO-HP 17.76mg and DSPE-mPEG200000.96mg were weighed respectively, added with 2mL of anhydrous ethanol, heated to 65℃ to dissolve, as the oil phase, 400mM sucrose octasulfate triethylamine 6mL preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (21mL / min:7mL / min), and the organic solvent was removed by reduced pressure evaporation to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration to obtain irinotecan hydrochloride blank liposomes. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are then stored in a 4°C refrigerator.

[0056] Comparative Example 13

[0057] 54.48 mg of DSPC, 17.76 mg of CHO-HP and 0.96 mg of DSPE-mPEG2000 were weighed, 2 mL of anhydrous ethanol was added, and the mixture was heated to 65 °C to dissolve as the oil phase. 6 mL of 400 mM sucrose octasulfate triethylamine was preheated at 65 °C as the water phase. The reaction pipeline and microchannel reactor were placed in a 60 °C environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (42 mL / min: 14 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 to obtain blank irinotecan hydrochloride liposomes. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are then stored in a 4°C refrigerator.

[0058] Comparative Example 14

[0059] 54.48 mg of DSPC, 17.76 mg of CHO-HP and 0.96 mg of DSPE-mPEG2000 were weighed, 2 mL of anhydrous ethanol was added, and the mixture was heated to 65 °C to dissolve as the oil phase. 6 mL of 400 mM sucrose octasulfate triethylamine was preheated at 65 °C as the water phase. The reaction pipeline and microchannel reactor were placed in a 60 °C environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (66 mL / min: 22 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 to obtain blank irinotecan hydrochloride liposomes. Weigh 17.2 mg of irinotecan hydrochloride trihydrate, dissolve it in 1 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are then stored in a 4°C refrigerator.

[0060] Comparative Example 15

[0061] DSPC 45.97mg, CHO-HP 15.99mg and DSPE-mPEG200000.81mg were weighed respectively, 3mL of anhydrous ethanol was added, and the mixture was heated to 65℃ to dissolve as the oil phase. 225mM sucrose octasulfate triethylamine 9mL was preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (66mL / min:22mL / min). The organic solvent was removed by evaporation under reduced pressure to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0062] Comparative Example 16

[0063] DSPC 45.97mg, CHO-HP 15.99mg and DSPE-mPEG200000.81mg were weighed respectively, added with 3mL of anhydrous ethanol, heated to 65℃ to dissolve, as the oil phase, 165mM sucrose octasulfate triethylamine 9mL preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (66mL / min:22mL / min), and the organic solvent was removed by reduced pressure evaporation to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0064] Comparative Example 17

[0065] DSPC 45.97mg, CHO-HP 15.99mg and DSPE-mPEG200000.81mg were weighed respectively, added with 3mL of anhydrous ethanol, heated to 65℃ to dissolve, as the oil phase, 140mM sucrose octasulfate triethylamine 9mL preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (66mL / min:22mL / min), and the organic solvent was removed by reduced pressure evaporation to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0066] Comparative Example 18

[0067] DSPC 45.97mg, CHO-HP 15.99mg and DSPE-mPEG200000.81mg were weighed respectively, added with 3mL of anhydrous ethanol, heated to 65℃ to dissolve, as the oil phase, 94mM sucrose octasulfate triethylamine 9mL preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (66mL / min:22mL / min), and the organic solvent was removed by reduced pressure evaporation to obtain a liposome suspension, which was incubated for 20min. The free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0068] Comparative Example 19

[0069] DSPC 40.86mg, CHO-HP 13.32mg and DSPE-mPEG200000.72mg were weighed respectively, added with 2.4mL of anhydrous ethanol, heated to 65℃ to dissolve, as the oil phase, 140mM sucrose octasulfate triethylamine 12mL preheated at 65℃ as the water phase. The reaction pipeline and microchannel reactor were placed in a 60℃ environment for heat preservation, and the water and oil phases were mixed through the microchannel reactor at a flow rate ratio of (50mL / min:10mL / min), and the organic solvent was removed by reduced pressure evaporation to obtain a liposome suspension, which was incubated for 20min. 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. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0070] Comparative Example 20

[0071] 54.48 mg of DSPC, 17.76 mg of CHO-HP and 0.96 mg of DSPE-mPEG2000 were weighed respectively, 2.4 mL of anhydrous ethanol was added, and the mixture was heated to 65 °C to dissolve as the oil phase. 12 mL of 140 mM sucrose octasulfate triethylamine was preheated at 65 °C as the water phase. The reaction pipeline and microchannel reactor were placed in a 60 °C environment for heat preservation, 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 free sucrose octasulfate triethylamine outside the liposome was removed by membrane filtration, and the liposome was concentrated to 1 / 2 times the original volume to obtain irinotecan hydrochloride blank liposomes. Weigh 24.51 mg of irinotecan hydrochloride trihydrate, dissolve it in 1.9 mL of HEPES buffer, and slowly add it dropwise 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 to obtain irinotecan hydrochloride liposomes, which are stored in a 4°C refrigerator.

[0072] Test Example 1: Testing liposome particle size

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

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

[0075]

[0076]

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

[0078] The results show that the particle size of the irinotecan hydrochloride liposomes prepared in Examples 1 to 3 is controlled between 90-115 nm, and the 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, with excellent monodispersity and uniform particle size distribution, which helps to prolong the retention time of the irinotecan hydrochloride liposomes in tumor tissues, thereby better exerting the anti-tumor effect. Among them, the TEM (transmission electron microscope) image of the irinotecan hydrochloride liposomes prepared in Example 3 is as follows Figure 1 As shown, the liposomes have regular morphology, uniform size, and a diameter of about 100 nm.

[0079] Comparative Examples 1 to 9 added the oil phase to the aqueous phase by dropwise addition, and used ultrasonic fragmentation 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, the drug-lipid ratio and other parameters, only Comparative Example 3 achieved a good particle size and distribution, and the other samples could not produce irinotecan hydrochloride liposomes with a particle size between 90-115 nm and a PDI less than 0.2.

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

[0081] Test Example 2: Testing liposome encapsulation efficiency

[0082] Take 1 ml of irinotecan hydrochloride liposomes prepared in Examples 1-3 and Comparative Examples 1-20 respectively in a dialysis bag, put 600 ml of water outside, dialyze for 6 hours under stirring, take the dialysate and use HPLC to determine the content of irinotecan hydrochloride liposomes to make W. Take another 1 ml of irinotecan hydrochloride liposomes in a 100 ml volumetric flask, make the volume with methanol, ultrasonically break the emulsion, analyze by HPLC, and make W. The encapsulation efficiency formula is: EE = (W total - W flow) / W total × 100%.

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

[0084]

[0085]

[0086] In Examples 1 to 3, microfluidic devices were used to prepare irinotecan hydrochloride liposomes, and the oil-water 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, and the DSPE-mPEG2000 concentration was 0.45 mg / mL. The prepared irinotecan hydrochloride liposomes can achieve a high encapsulation rate of up to 95%.

[0087] In particular, by using the formulation of Example 3, after the oil-water phases are mixed by a microfluidic device, the blank liposomes are incubated for only 5 minutes, and then the ethanol and the free inner water phase are directly removed by membrane packaging to obtain irinotecan hydrochloride liposomes with an encapsulation rate of more than 95%. Therefore, the process of Example 3 is simpler in actual implementation and has significant advantages in process scale-up.

[0088] Comparative Examples 1 to 9 use an 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 sucrose octasulfate triethylamine, the amount of sucrose octasulfate triethylamine, the incubation time, the incubation temperature, the amount of irinotecan hydrochloride, the drug-lipid ratio and other parameters, except for the relatively ideal encapsulation rate of Comparative Example 3, the encapsulation rate of liposomes prepared in other cases is not high, and it is generally difficult to reach 90%. Comparative Examples 10 to 20 use methods similar to those of Examples 1 to 3, but due to the change in the prescription, the encapsulation rate is generally not high.

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

[0090] 1 mL of the liposome of Example 3 was placed in a 8000-14000Da dialysis bag, sealed, and 100 mL of release solution was poured in, the release solution being a PBS solution of pH 6.5 containing 10 mM ammonium sulfate, and the solution was shaken in a 37°C water bath constant temperature oscillator. Part of the release solution was taken out at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and fresh release medium was replaced. The released solution was analyzed by HPLC to calculate the cumulative release percentage.

[0091] like Figure 2 As shown, irinotecan hydrochloride liposomes are slowly released in the release medium, and about 50% is released in 24 hours. Sucrose octasulfate and irinotecan hydrochloride can form precipitation in the inner aqueous phase, which improves the retention effect of irinotecan hydrochloride in the liposomes, significantly slows down the drug release rate, and has an excellent sustained release effect.

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

[0093] The liposomes of the example and the liposomes of comparative example 3 were placed in an environment of 2-8° C., and the physical states of the samples were observed regularly to examine the product stability. The results are shown in Table 3.

[0094] Table 3 Product stability records

[0095]

[0096] The results show that the irinotecan hydrochloride liposome preparation of the embodiment maintains good stability throughout the 6-month observation period, always maintaining the state of a light yellow translucent suspension, without obvious physical property changes or precipitation. This shows that the liposomes prepared by microfluidic equipment and strictly controlling the oil-water ratio, initial lipid concentration and other parameters have excellent long-term stability. In contrast, although the liposomes of Comparative Example 3 also appear as a light yellow translucent suspension at the beginning, a very small amount of precipitation occurs after 1 month of storage, and as time extends to 3 months and 6 months, the precipitation phenomenon is more obvious. This shows that although Comparative Example 3 achieves a good particle size and distribution, its overall formula and / or preparation method are still insufficient, and the long-term stability of irinotecan hydrochloride liposomes cannot be achieved.

[0097] The above embodiments are exemplary, and their purpose is to illustrate the technical concept and features of the present invention so that people familiar with the technology in this field can understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for preparing irinotecan hydrochloride liposomes, characterized in that: The following steps are involved: 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 a liposome material in an organic solvent as an oil phase, wherein the total lipid concentration is 22-34 mg / mL; mixing the water-oil phases by a microfluidic device to prepare blank liposomes; Step S2: removing free sucrose octasulfate triethylamine and organic solvent outside the blank liposome; Step S3: Dissolve irinotecan hydrochloride trihydrate in HEPES buffer, add it into blank liposomes at a drug-lipid volume ratio of 1:1-1:3 for active drug loading, and incubate at 50-70° C. to obtain irinotecan hydrochloride liposomes.

2. The process for preparing irinotecan hydrochloride liposomes according to claim 1, characterized in that: In step S1, the concentration of sucrose octasulfate triethylamine in the aqueous phase is 140-150 mM.

3. The process for preparing irinotecan hydrochloride liposomes according to claim 2, characterized in that: In step S1, the liposome material is dissolved in ethanol to obtain an oil phase; the liposome material includes distearoylphosphatidylcholine (DSPC), cholesterol hemisuccinate hydrogenated vegetable oil ester (CHO-HP), and N-(carbonyl monomethoxy polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-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.

4. The process for preparing irinotecan hydrochloride liposomes according to claim 1, characterized in that: In step S1, water and oil are mixed by a microfluidic device, and the flow ratio of the oil and water phases is controlled to be 1:(5±0.3).

5. The process for preparing irinotecan hydrochloride liposomes according to claim 1, characterized in that: In step S1, the oil-water two-phase is mixed at 50-70°C and incubated for 5-60 minutes after mixing.

6. The process for preparing irinotecan hydrochloride liposomes according to any one of claims 1 to 5, characterized in that: In step S2, HEPES buffer at pH 6.0-8.0 is used as a replacement medium, and 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.

7. The process for preparing irinotecan hydrochloride liposomes according to claim 6, characterized in that: In step S2, the molecular weight cut-off of the tangential flow filtration membrane package device is 100 kd.

8. The process for preparing irinotecan hydrochloride liposomes according to claim 6, characterized in that: In step S3, the concentration of irinotecan hydrochloride trihydrate is 2.0-30.0 mg / mL.

9. An irinotecan hydrochloride liposome, characterized in that: The irinotecan hydrochloride liposomes are prepared according to the preparation process of any one of claims 1 to 8; the particle size of the irinotecan hydrochloride liposomes is 90-115 nm, the dispersity index is less than 0.2, and the encapsulation rate is ≥95%.

10. Use of the preparation process of irinotecan hydrochloride liposome according to any one of claims 1 to 8 in the preparation of irinotecan hydrochloride liposome injection.

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