Doxorubicin hydrochloride liposome, preparation and application thereof

Abstract

The invention discloses a doxorubicin hydrochloride liposome, a preparation and application thereof, and belongs to the technical field of antitumor drug preparations. The doxorubicin hydrochloride liposome disclosed by the invention is prepared from doxorubicin hydrochloride, phospholipid, cholesterol, beta-sitosterol, cucumber alcohol, glyceryl behenate and a pH buffer agent as raw materials through a specific preparation method. The components in the formula have a synergistic effect to stabilize the lipid membrane, the preparation process is simple, and industrialization is easy to realize. Experiments show that the liposome is high in encapsulation efficiency, and the injection prepared from the liposome is better than commercially available products in stability and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparations, and particularly relates to a doxorubicin hydrochloride liposome, a preparation thereof and an application thereof. Background Art

[0002] Doxorubicin is a widely used anti-tumor drug, mainly used for the treatment of various malignant tumors, such as breast cancer, leukemia, lymphoma, etc. It exerts its anti-cancer effect by interfering with the DNA synthesis and replication of tumor cells. However, doxorubicin often faces some significant problems in clinical applications, such as its low solubility and non-specific toxicity, which lead to its high side effects and low bioavailability. In addition, the anti-tumor effect of doxorubicin is also affected by its rapid clearance and uneven distribution in the body.

[0003] To solve these problems, liposomes, as a drug delivery system, have been widely studied and applied. Liposomes can effectively encapsulate and carry drugs, improve the solubility of drugs, and reduce their toxic side effects by improving the pharmacokinetic properties of drugs.

[0004] Chinese Patent with publication number CN1018977667A discloses a doxorubicin hydrochloride liposome injection and its preparation process, including doxorubicin hydrochloride, hydrogenated soy lecithin, cholesterol, polyethylene glycolated lipid, organic acid or ammonium sulfate, sugar, buffer, and water for injection. It is prepared by lipid phase freeze-drying, lipid phase hydration, liposome sizing, creating a transmembrane gradient inside and outside the phospholipid membrane, liposome drug loading, sterilization, and dispensing. However, its preparation method is relatively complex, the entrapment efficiency data is doubtful, the stability is not verified, and industrial production cannot be achieved.

[0005] Chinese Patent with publication number CN 118986885 A discloses a highly stable doxorubicin liposome injection preparation and its preparation process. The steps include: S1: preparing doxorubicin liposome: S11: preparing distearoyl phosphatidylethanolamine-polyethylene glycol-tocopherol; S12: shear-mixing hydrogenated lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol-tocopherol, cholesterol, absolute ethanol, and ammonium sulfate, filtering to obtain a crude blank liposome; S13: performing dialysis treatment on the crude blank liposome to obtain a blank liposome suspension; S14: adding a doxorubicin solution to the blank liposome suspension, heating and keeping warm, and then performing sterilization treatment and rotary evaporation under reduced pressure to obtain doxorubicin lipid; S2: formulating the doxorubicin liposome and compound water for injection into a highly stable doxorubicin liposome injection preparation for storage and standby. There are also problems of complex process, and the entrapment efficiency is not verified. Using only the concentration change of doxorubicin liposome as the basis for stability verification is not comprehensive enough.

[0006] Doxorubicin hydrochloride liposomes in the prior art still have problems such as low encapsulation efficiency, poor stability, and complex preparation processes, which will directly affect their anti-tumor effects. In addition, due to the poor stability of liposomes, the drug content is unstable, affecting the therapeutic effect. Therefore, there is an urgent need for a new type of doxorubicin liposome and its preparation that can not only improve the encapsulation efficiency of doxorubicin hydrochloride, improve the stability of liposomes, but also have a simple preparation process and are easy to industrialize. Summary of the Invention

[0007] The present invention aims to solve the problems of low encapsulation efficiency, poor stability, and complex preparation process of existing doxorubicin hydrochloride liposomes. A doxorubicin hydrochloride liposome is provided, which includes 10 parts of doxorubicin hydrochloride, 10 - 20 parts of phospholipid, 1 - 3 parts of cholesterol, 1 - 3 parts of β-sitosterol (CAS: 83-46-5), 0.5 - 1.5 parts of cucurbitol (CAS: 7786-44-9), 0.2 - 0.8 parts of glyceryl behenate, and a pH buffer in parts by weight. Among them, the pH buffer is selected from at least one of histidine, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate, and disodium hydrogen phosphate; the phospholipid is preferably hydrogenated soy lecithin and phosphatidylserine, and the mass ratio is 1:1 - 3:1; the pH value of the pH buffer is controlled at 5.5 - 6.5.

[0008] In the formation process of liposomes, each component plays a synergistic role. Phospholipids, as the main components of the liposome membrane, form a bilayer structure, providing a basic framework for drug encapsulation. Cholesterol can regulate the fluidity and stability of the lipid membrane and enhance the rigidity of the membrane. The addition of β-sitosterol, cucurbitol, and glyceryl behenate can interact with phospholipids and cholesterol to further stabilize the lipid membrane structure.

[0009] Its preparation method includes: dissolving cholesterol, β-sitosterol, cucurbitol, and glyceryl behenate in chloroform and ethanol with a volume ratio of 3 - 4:1, and performing ultrasonic treatment at 2 - 8°C for 10 - 30 min to obtain a cold solution; dissolving phospholipids in chloroform, removing 1 / 3 - 1 / 2 of the solvent by rotary evaporation under reduced pressure at 40 - 55°C, quickly adding the cold solution, and then removing the solvent by rotary evaporation under reduced pressure at 50 - 70°C to obtain a thin film; finally, dissolving doxorubicin hydrochloride in a pH buffer and adding it to the thin film for hydration, and obtaining the product through ultrasonic treatment and high-pressure homogenization. Stepwise dissolution and low-temperature ultrasonic treatment can enable lipid molecules to be arranged orderly in a low-temperature environment, avoiding disordered movement of molecules due to too high temperature, thereby ensuring the stability and uniformity of the lipid membrane structure and further enhancing the encapsulation stability.

[0010] In addition, the application of the liposome in preparing an anti-tumor drug (such as doxorubicin hydrochloride liposome injection) is also provided. The injection consists of doxorubicin hydrochloride liposome and at least one lyoprotectant selected from sucrose, mannitol, and lactose. During the application process, the liposome can effectively improve the stability of doxorubicin hydrochloride and improve the bioavailability of the drug.

[0011] Compared with the prior art, the present invention has the following remarkable technical advantages:

[0012] (1) High encapsulation rate: Through unique formulation design and preparation process, the encapsulation rate of doxorubicin hydrochloride liposomes prepared by the present invention is significantly improved. In the accelerated tests at high temperature and high humidity (40°C / 75% RH) in Examples 1-3, the encapsulation rate on the 0th day was above 93%, and the decline rate of the encapsulation rate within 180 days was less than 1.5%, which is significantly higher than that of Comparative Examples 1-6.

[0013] (2) Good stability: The doxorubicin hydrochloride liposomes and their injection solutions of the present invention have excellent stability. From the comparative experiments of the doxorubicin hydrochloride liposome injection solutions prepared in Examples 4-6 and the commercially available products, it can be seen that under the same conditions of high temperature and high humidity (40°C / 75% RH), the change in the content of doxorubicin in the products of the present invention is smaller within 180 days, and the stability is better than that of the commercially available products.

[0014] (3) Simple process and easy industrialization: The preparation process of the present invention adopts conventional operations such as dissolution, ultrasonic treatment, rotary evaporation under reduced pressure, and high-pressure homogenization, without complex steps and special equipment, is easy to operate, and is easy to realize industrial production, reducing the production cost and production difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Encapsulation rates of doxorubicin hydrochloride liposomes in Examples 1-3 and doxorubicin hydrochloride liposomes in Comparative Examples 1-6.

[0016] Figure 2 : Content changes of doxorubicin hydrochloride liposome injection solutions in Examples 4-6 and commercially available doxorubicin hydrochloride liposome injection solutions. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] In order to make the objectives and technical solutions of the present invention clearer, the following further describes the present invention with reference to the embodiments. However, the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. Those skilled in the art should understand that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0018] Doxorubicin Hydrochloride Liposomes in Example 1

[0019] Formulation:

[0020]

[0021]

[0022] Preparation method:

[0023] (1) Dissolve cholesterol, β-sitosterol, cucurbitol, and behenyl glycerol in 50 ml of a 3:1 chloroform and ethanol volume ratio, and sonicate for 20 min at 5 °C to obtain a cold solution;

[0024] (2) Dissolve phospholipids in 100 ml of chloroform, and rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed. Quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent and obtain a film;

[0025] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and perform high-pressure homogenization (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0026] Example 2 Doxorubicin Hydrochloride Liposomes

[0027] Formulation:

[0028]

[0029] Preparation method:

[0030] (1) Dissolve cholesterol, β-sitosterol, cucurbitol, and behenyl glycerol in 40 ml of a 3:1 chloroform and ethanol volume ratio, and sonicate for 10 min at 2 °C to obtain a cold solution;

[0031] (2) Dissolve phospholipids in 80 ml of chloroform, and rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed. Quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent and obtain a film;

[0032] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and perform high-pressure homogenization (pressure 1000 bar, 3 cycles, flow rate 5 ml / min, temperature 45 °C) to obtain the product.

[0033] Example 3 Doxorubicin Hydrochloride Liposomes

[0034] Formulation:

[0035]

[0036] Preparation method:

[0037] (1) Dissolve cholesterol, β-sitosterol, cucurbitol, and behenyl glycerol in 80 ml of a 4:1 chloroform and ethanol volume ratio, and sonicate for 30 min at 8 °C to obtain a cold solution;

[0038] (2) Dissolve the phospholipid in 120 ml of chloroform, and rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed. Quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent, obtaining a thin film;

[0039] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the thin film for hydration, sonicate for 10 min, and homogenize under high pressure (pressure 2000 bar, circulate 5 times, flow rate 15 ml / min, temperature 55 °C) to obtain the product.

[0040] Comparative Example 1 Doxorubicin Hydrochloride Liposome

[0041] Formula:

[0042]

[0043]

[0044] Preparation method:

[0045] (1) Dissolve cholesterol, cucurbitol, and glyceryl behenate in 50 ml of a 3:1 chloroform and ethanol volume ratio, and sonicate at 5 °C for 20 min to obtain a cold solution;

[0046] (2) Dissolve the phospholipid in 100 ml of chloroform, and rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed. Quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent, obtaining a thin film;

[0047] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the thin film for hydration, sonicate for 10 min, and homogenize under high pressure (pressure 15 bar, circulate 4 times, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0048] Comparative Example 2 Doxorubicin Hydrochloride Liposome

[0049] Formula:

[0050]

[0051] Preparation method:

[0052] (1) Dissolve cholesterol, β-sitosterol, and glyceryl behenate in 50 ml of a 3:1 chloroform and ethanol volume ratio, and sonicate at 5 °C for 20 min to obtain a cold solution;

[0053] (2) Dissolve the phospholipid in 100 ml of chloroform, and rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed. Quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent, obtaining a thin film;

[0054] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and homogenize under high pressure (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0055] Control Example 3 Doxorubicin Hydrochloride Liposome

[0056] Formulation:

[0057]

[0058] Preparation method:

[0059] (1) Dissolve cholesterol, β-sitosterol, and cucurbitol in 50 ml of chloroform and ethanol with a volume ratio of 3:1, sonicate at 5 °C for 20 min to obtain a cold solution;

[0060] (2) Dissolve phospholipids in 100 ml of chloroform, rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed, quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent to obtain a film;

[0061] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and homogenize under high pressure (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0062] Control Example 4 Doxorubicin Hydrochloride Liposome

[0063] Formulation:

[0064]

[0065] Preparation method:

[0066] (1) Dissolve cholesterol, stigmasterol, cucurbitol, and glyceryl behenate in 50 ml of chloroform and ethanol with a volume ratio of 3:1, sonicate at 5 °C for 20 min to obtain a cold solution;

[0067] (2) Dissolve phospholipids in 100 ml of chloroform, rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed, quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent to obtain a film;

[0068] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and homogenize under high pressure (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0069] Control Example 5 Doxorubicin Hydrochloride Liposome

[0070] Formulation:

[0071]

[0072] Preparation method:

[0073] (1) Dissolve cholesterol, β-sitosterol, cucurbitol, glyceryl behenate, and phospholipids in 150 ml of chloroform, and remove the solvent by rotary evaporation under reduced pressure at 40 - 70 °C to obtain a film;

[0074] (2) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and perform high-pressure homogenization (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0075] Doxorubicin hydrochloride liposome of Comparative Example 6

[0076] Formulation:

[0077]

[0078]

[0079] Preparation method:

[0080] (1) Dissolve cholesterol, β-sitosterol, cucurbitol, glyceryl behenate in 50 ml of a 3:1 volume ratio of chloroform and ethanol to obtain Solution A;

[0081] (2) Dissolve phospholipids in 100 ml of chloroform, rotary evaporate under reduced pressure at 40 - 55 °C until 1 / 3 - 1 / 2 of the solvent is removed, quickly add Solution A, and rotary evaporate under reduced pressure at 50 - 70 °C to remove the solvent to obtain a film;

[0082] (3) Dissolve doxorubicin hydrochloride in 50 ml of pH buffer, add it to the film for hydration, sonicate for 10 min, and perform high-pressure homogenization (pressure 15 bar, 4 cycles, flow rate 10 ml / min, temperature 50 °C) to obtain the product.

[0083] Entrapment efficiency of doxorubicin hydrochloride liposome

[0084] The entrapment efficiency of liposome refers to the proportion of the drug encapsulated in the liposome, which reflects the effective loading capacity of the carrier and the preparation process for the drug. Its core index is the percentage of the encapsulated drug in the total drug dosage, which is a key parameter for evaluating the quality of liposome drugs. Place the doxorubicin hydrochloride liposomes of Examples 1 - 3 and the doxorubicin hydrochloride liposomes of Comparative Examples 1 - 6 at a temperature of 40 ± 2 °C and a relative humidity of 75% ± 5%. Sampling is carried out on the 0th day, 30th day, 90th day, and 180th day, and the entrapment efficiency of the doxorubicin hydrochloride liposome is measured by the ultrafiltration centrifugation method and the HPLC detection method.

[0085] Table 1 Entrapment efficiency of doxorubicin hydrochloride liposome

[0086]

[0087]

[0088] Table 1 shows that the encapsulation efficiency of doxorubicin hydrochloride liposomes in Examples 1-3 was significantly higher than that of doxorubicin hydrochloride liposomes in Comparative Examples 1-6 in the accelerated test under high temperature and high humidity (40°C / 75% RH), and the stability was better. The decline in the encapsulation efficiency within 180 days was less than 1.5%. This indicates that cholesterol, β-sitosterol, cucurbitol, and glyceryl behenate can synergistically stabilize the lipid membrane, which is more conducive to encapsulating doxorubicin hydrochloride. The stepwise dissolution and low-temperature ultrasonic treatment in the preparation process can ensure the orderly arrangement of lipid molecules and improve the encapsulation stability.

[0089] Doxorubicin Hydrochloride Liposome Injection of Example 4

[0090] The doxorubicin hydrochloride liposome of Example 1 was added with 2.5 g of sucrose and freeze-dried to obtain the product.

[0091] Doxorubicin Hydrochloride Liposome Injection of Example 5

[0092] The doxorubicin hydrochloride liposome of Example 2 was added with 1 g of mannitol and freeze-dried to obtain the product.

[0093] Doxorubicin Hydrochloride Liposome Injection of Example 6

[0094] The doxorubicin hydrochloride liposome of Example 3 was added with 5 g of lactose and freeze-dried to obtain the product.

[0095] Commercially available doxorubicin hydrochloride liposome injection (H20110209)

[0096] Stability of Doxorubicin Hydrochloride Liposome Injection

[0097] The doxorubicin hydrochloride liposome injections of Examples 4-6 and the commercially available doxorubicin hydrochloride liposome injection were placed at a temperature of 40 ± 2°C and a relative humidity of 75% ± 5%. Samples were taken on the 0th day, 30th day, 90th day, and 180th day, and the content of doxorubicin was determined by the high performance liquid chromatography method (General Rule 0512 of the Chinese Pharmacopoeia).

[0098] Test solution: Weigh an appropriate amount of this product accurately, dissolve it in the mobile phase and quantitatively dilute to prepare a solution containing about 0.1 mg per 1 ml. Reference solution: Weigh doxorubicin hydrochloride reference substance accurately, dissolve it in the mobile phase and quantitatively dilute to prepare a solution containing about 0.1 mg per 1 ml. System suitability solution: Weigh appropriate amounts of doxorubicin hydrochloride reference substance and epirubicin hydrochloride reference substance, dissolve them in the mobile phase and dilute to prepare a mixed solution containing 50 μg of each per 1 ml. Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler; use sodium dodecyl sulfate solution (weigh 1.44 g of sodium dodecyl sulfate and 0.68 ml of phosphoric acid, add water to 500 ml to dissolve)-acetonitrile-methanol (500:500:60) as the mobile phase; the detection wavelength is 254 nm; the injection volume is 10 μl. System suitability requirements: In the chromatogram of the system suitability solution, the resolution between the doxorubicin peak and the epirubicin peak should be greater than 2.0. Assay method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate by the external standard method based on the peak area.

[0099] Figure 2 For the content changes of doxorubicin hydrochloride liposome injection in Examples 4 to 6 and the commercially available doxorubicin hydrochloride liposome injection, the data show that the stability of the doxorubicin hydrochloride liposome injection in Examples 4 to 6 of the present invention is better than that of the commercially available doxorubicin hydrochloride liposome injection.

Claims

1. A liposomal doxorubicin hydrochloride, characterized in that, The doxorubicin hydrochloride liposome comprises, by weight parts, 10 parts of doxorubicin hydrochloride, 10 - 20 parts of phospholipid, 1 - 3 parts of cholesterol, 1 - 3 parts of β-sitosterol, 0.5 - 1.5 parts of cucurbitol, 0.2 - 0.8 parts of glyceryl behenate, and a pH buffer.

2. The liposomal doxorubicin hydrochloride according to claim 1, wherein The pH buffer is selected from at least one of histidine, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

3. The doxorubicin hydrochloride liposome according to claim 1, characterized in that, The phospholipid is hydrogenated soy lecithin, phosphatidylserine; preferably, the mass ratio of hydrogenated soy lecithin to phosphatidylserine is 1:1 - 3:

1.

4. The doxorubicin hydrochloride liposome according to claim 1, wherein, The pH of the pH buffer is 5.5 - 6.

5.

5. A method for preparing the liposome doxorubicin hydrochloride according to claim 1, characterized in that, The method comprises the following steps: (1) Dissolve cholesterol, β-sitosterol, cucurbitol, and glyceryl behenate in organic solvent A, and ultrasonicate at 2 - 8°C for 10 - 30 min to obtain a cold solution; (2) Dissolve the phospholipid in organic solvent B, and rotary evaporate under reduced pressure at 40 - 55°C until 1 / 3 - 1 / 2 of the solvent is removed, quickly add the cold solution, and rotary evaporate under reduced pressure at 50 - 70°C to remove the solvent to obtain a film; (3) Dissolve doxorubicin hydrochloride in the pH buffer, add it to the film for hydration, ultrasonicate, and high-pressure homogenize to obtain the product.

6. The method according to claim 5, wherein The organic solvent A is a mixture of chloroform and ethanol with a volume ratio of 3 - 4:1, and the organic solvent B is chloroform.

7. Use of the doxorubicin hydrochloride liposome according to claim 1 in the preparation of an anti-tumor drug.

8. According to the use of claim 7, the anti-tumor drug is a doxorubicin hydrochloride liposome injection.

9. According to the use of claim 8, the doxorubicin hydrochloride liposome injection is composed of a doxorubicin hydrochloride liposome and a lyoprotectant.

10. According to the use of claim 9, the lyoprotectant is selected from at least one of sucrose, mannitol, and lactose.

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