Topotecan liposome and preparation method and application thereof

Abstract

The application discloses a topotecan liposome and a preparation method and application thereof, which is prepared from raw materials including a topotecan pharmaceutically acceptable salt, cholesterol, phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropyl ammonium chloride, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol, the topotecan liposome of the application is uniform in shape after being placed for 30 days, and no precipitation is separated out, so that the stability of the topotecan liposome is improved. The topotecan liposome can provide strong targeting liver, spleen, lung, kidney and other organs, so that the accumulation amount of the topotecan at a tumor site is significantly improved, thereby enhancing the drug treatment effect and widening the application of the topotecan in treating cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, the present invention relates to a topotecan liposome, as well as a preparation method and application thereof. Background Art

[0002] Topotecan (Topotecan, with the structural formula being with the English trade name Hycamtin and Chinese trade name He Meixin) is a semi-synthetic novel antitumor drug launched on the US market by Smithkline Beecham Pharmaceuticals in 1996, and is also the world's first antitumor drug acting on topoisomerase I. At present, it is widely used in patients with metastatic ovarian cancer who have failed initial chemotherapy or sequential chemotherapy, and patients with small cell lung cancer who are sensitive to chemotherapy and have failed first-line chemotherapy.

[0003] Topotecan exerts its cytotoxic effect by inhibiting topoisomerase I (Topoisomerase I, Topo I). Since the reverse rotation of DNA during replication will cause tangles, positive and negative supercoils, etc., to ensure the normal progress of replication, the participation of Topo must be relied on. Topo can perform DNA cleavage, rotation and re-ligation, so that DNA can smoothly perform unwinding, replication, transcription, etc. Topotecan embeds into the cleaved complex of DNA-Topo I to form a "drug-Topo I-DNA" ternary complex. This complex is not toxic, but can inhibit the repair of DNA nicks. In the ternary complex, topotecan increases the distance between base pairs at the break site, becoming an obstacle to the closure of the DNA strand. When the moving replication fork encounters the ternary complex during DNA replication, it will cause irreversible double-strand breaks of DNA, and the broken DNA will continuously accumulate in the cell, thereby leading to cell death. Compared with normal cells, the content and activity of Topo I in tumor cells are significantly increased. Therefore, drugs that inhibit Topo I affect the growth of tumor cells by inhibiting DNA replication in tumor cells.

[0004] There have been some reports on topotecan / topotecan hydrochloride liposomes, which to a certain extent reduce the side effects of topotecan and improve the therapeutic effect. However, the inventors of the present invention have found that the stability of the existing topotecan / topotecan hydrochloride liposomes is relatively poor, the drug is prone to precipitate after being placed for a period of time, which limits the development of liposomes. In addition, the tumor targeting property of topotecan liposomes is not strong, and due to the fast metabolic rate, they cannot be targeted and enriched at the tumor site for a long time. Summary of the Invention

[0005] The objective of the present invention is to improve the stability of topotecan liposomes and enhance their tumor targeting property at the same time.

[0006] The specific technical solutions for achieving the above-mentioned objective of the present invention include the following contents.

[0007] A first aspect of the present invention provides a topotecan liposome, which is made from raw materials comprising the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–30 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned topotecan liposomes, comprising the following steps: (1) The cholesterol, phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol are dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve the pharmaceutically acceptable salt of the topotecan in buffer solution or sterile water for injection to obtain an aqueous phase; (3) Mix the lipid phase with the aqueous phase to obtain the final product.

[0009] A third aspect of the present invention provides the use of the above-described topotecan liposomes in the preparation of antitumor drugs. The inventors of this invention discovered that by using a lipid phase composed of specific types of cationic lipids, zwitterionic lipids, and neutral lipids as a carrier to encapsulate pharmaceutically acceptable salts of topotecan, and by controlling the amount of drug and lipid phase within a suitable range, topotecan liposomes were prepared. After being stored for 30 days, the liposomes exhibited uniform properties, no precipitation, and significantly improved stability.

[0010] When the topotecan liposomes prepared in this invention are applied to tumor-bearing mice, they can provide a strong ability to target organs such as the liver, spleen, lungs, and kidneys, significantly increasing the accumulation of topotecan at the tumor site, thereby enhancing the therapeutic effect and broadening the application of topotecan in the treatment of cancer. Attached Figure Description

[0011] Fig. 1 The images show the properties of liposome samples 1, 5, 8, and 11 at day 0.

[0012] Fig. 2 The images show the properties of liposome samples 1, 5, 8, and 11 of this invention after being placed at 2-8°C for 10 days.

[0013] Fig. 3 The images show the properties of liposome samples 1, 5, and 11 of this invention after being stored at 2-8°C for 30 days.

[0014] Fig. 4 This is a comparison chart showing the accumulation of liposome samples 5 and 6 in the lungs at 1h, 4h, and 24h in Test Example 2 of the present invention.

[0015] Fig. 5 The images show in vivo images (from left to right) of the heart, liver, spleen, lungs, and kidneys of mice at 1h, 4h, and 24h after administration of the liposome sample 5 in Experiment Example 2 of this invention.

[0016] Fig. 6 The images show in vivo images (from left to right) of the heart, liver, spleen, lungs, and kidneys of mice at 1h, 4h, and 24h after administration of liposome sample 6 in Experiment Example 2 of this invention. Detailed Implementation

[0017] To facilitate understanding of the present invention, a more comprehensive description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0019] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0020] In some embodiments of the present invention, a topotecan liposome is disclosed, which is made from raw materials comprising the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–30 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

[0021] In one embodiment, the topotecan liposomes are made from raw materials comprising the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–20 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

[0022] In one embodiment, the topotecan liposomes are made from raw materials comprising the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–15 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

[0023] In one embodiment, the topotecan liposomes are made from raw materials comprising the following concentrations: Pharmaceutically acceptable salt of topotecan 12 mg / mL Cholesterol 12.14 mg / mL Phosphatidylcholine 95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2.12 mg / mL.

[0024] In one embodiment, the pharmaceutically acceptable salt of topotecan is topotecan hydrochloride.

[0025] In other embodiments of the present invention, a method for preparing the above-mentioned topotecan liposomes is disclosed, comprising the following steps: (1) The cholesterol, phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol are dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve the pharmaceutically acceptable salt of the topotecan in buffer solution or sterile water for injection to obtain an aqueous phase; (3) Mix the lipid phase with the aqueous phase to obtain the final product.

[0026] In one embodiment, the organic solvent in step (1) is anhydrous ethanol.

[0027] In one embodiment, the buffer solution in step (2) is a PBS buffer solution.

[0028] In one embodiment, in step (3), the lipid phase and the aqueous phase are mixed at a total flow rate of 18 mL / min to 22 mL / min and a lipid phase:water phase flow rate ratio of 0.8 to 1.2:3.

[0029] In other embodiments of the present invention, the use of the above-mentioned topotecan liposomes in the preparation of antitumor drugs is disclosed.

[0030] In one embodiment, the tumor is metastatic ovarian cancer or small cell lung cancer.

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1 Topotecan liposomes and their preparation method In this embodiment, topotecan liposome sample 1 was prepared, and its formulation is shown in Table 1.

[0033]

[0034] Its preparation method includes the following steps: 1. Dissolve topotecan in PBS buffer to obtain the aqueous phase.

[0035] 2. Phosphatidylcholine, cholesterol, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol, and 1,2-dioleoyl-3-trimethylpropylammonium chloride were dissolved in anhydrous ethanol to obtain the lipid phase.

[0036] 3. Using a microfluidic device, the above lipid phase and aqueous phase are rapidly mixed at a total flow rate of 20 mL / min and a lipid (lipid phase) to water (aqueous phase) flow rate ratio of 1:3 to obtain the final product.

[0037] Example 2 Topotecan liposomes and their preparation method In this embodiment, topotecan liposome sample 2 was prepared, and its formulation is shown in Table 2.

[0038]

[0039] The preparation method is the same as in Example 1.

[0040] Example 3 Topotecan liposomes and their preparation method In this embodiment, topotecan liposome sample 3 was prepared, and its formulation is shown in Table 3.

[0041]

[0042] The preparation method is the same as in Example 1.

[0043] Example 4 Topotecan liposomes and their preparation method In this embodiment, topotecan liposome sample 4 was prepared, and its formulation is shown in Table 4.

[0044]

[0045] The preparation method is the same as in Example 1.

[0046] Example 5 Topotecan liposomes and their preparation method In this embodiment, topotecan liposome sample 5 was prepared, and its formulation is shown in Table 5.

[0047]

[0048] The preparation method is the same as in Example 1.

[0049] Comparative Example 1: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 6 was prepared in this comparative example, and its formulation is shown in Table 6.

[0050]

[0051] The preparation method is the same as in Example 1.

[0052] Comparative Example 2: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 7 was prepared in this comparative example, and its formulation is shown in Table 7.

[0053]

[0054] The preparation method is the same as in Example 1.

[0055] Comparative Example 3: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 8 was prepared in this comparative example, and its formulation is shown in Table 8.

[0056]

[0057] The preparation method is the same as in Example 1.

[0058] Comparative Example 4: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 9 was prepared in this comparative example, and its formulation is shown in Table 9.

[0059]

[0060] The preparation method is the same as in Example 1.

[0061] Comparative Example 5: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 10 was prepared in this comparative example, and its formulation is shown in Table 10.

[0062]

[0063] The preparation method is the same as in Example 1.

[0064] Comparative Example 6: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 11 was prepared in this comparative example, and its formulation is shown in Table 11.

[0065]

[0066] The preparation method is the same as in Example 1.

[0067] Comparative Example 7: Topotecan Liposomes and Their Preparation Method Topotecan liposome sample 12 was prepared in this comparative example, and its formulation is shown in Table 12.

[0068]

[0069] The preparation method is the same as in Example 1.

[0070] Experimental Example 1 The properties (after 30 days of storage at 2-8℃), particle size, zeta potential, and encapsulation efficiency of samples 1-12 of each embodiment and comparative example were measured, and the results are shown in Table 13. The properties of samples 1, 5, 8, and 11 after 0 days, 10 days, and 30 days of storage at 2-8℃ are as follows: Figs. 1-3 As shown.

[0071]

[0072] The results in Table 13 show that: The topotecan liposome samples 1-5 prepared in Examples 1-5 had small average particle size, positive zeta potential, and uniform properties after 30 days of storage, with no API precipitation, indicating sample stability. Among them, samples 1-2 and sample 5 had encapsulation efficiency exceeding 20%, indicating good encapsulation efficiency.

[0073] The topotecan liposome samples 6-12 prepared in Comparative Examples 1-7 also had small average particle sizes and positive zeta potentials. Except for sample 8, the encapsulation efficiency was very good. Except for sample 6, the stability of the other samples was poor, and API precipitated after standing. Among them, sample 10 precipitated before the aqueous and lipid phases were mixed.

[0074] The results of this experiment demonstrate that the topotecan liposomes prepared in this invention are highly stable within the liposomes.

[0075] Experimental Example 2 B16F10 tumor cells were used to establish a tumor model in C57BL / 6 mice. Mice bearing B16F10 tumors were selected and administered the sample 5 prepared in Example 5 and the sample 6 prepared in Comparative Example 1 (n=3) intravenously at a dose of 12 mg / kg. In vivo imaging was performed at 1 h, 4 h and 24 h after administration.

[0076] The cumulative results of liposome samples 5 and 6 in the lungs over time are shown in Table 14 and... Fig. 4 As shown. In vivo imaging images of liposome samples 5 and 6 in organs such as the heart, liver, spleen, lung, and kidney are shown below. Figs. 5-6 As shown.

[0077]

[0078] From Table 14 and Fig. 4 The results showed that at 1 h, 4 h, and 24 h after administration, the cumulative amount of sample 5 in the lungs was significantly higher than that of sample 6.

[0079] Fig. 5 and Fig. 6 The results showed that sample 5 also had significant targeting ability in organs such as the liver, spleen, and kidney, and its targeting ability was better than that of sample 6.

[0080] The results of this experiment show that the topotecan liposomes prepared in this invention can provide a strong ability to target organs such as the liver, spleen, lungs, and kidneys, allowing the drug to be further enriched at the tumor site, thereby enhancing the therapeutic effect and broadening the application of topotecan in the treatment of cancer.

[0081] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A topotecan liposome, characterized in that, It is made from raw materials containing the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–30 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

2. The topotecan liposome according to claim 1, characterized in that, It is made from raw materials containing the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–20 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

3. The topotecan liposome according to claim 2, characterized in that, It is made from raw materials containing the following concentrations: Pharmaceutically acceptable salt concentrations of topotecan are 10–15 mg / mL. Cholesterol 4.05~12.14 mg / mL Phosphatidylcholine 31.98~95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 8.10~24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 0.71~2.12 mg / mL.

4. The topotecan liposome according to claim 3, characterized in that, It is made from raw materials containing the following concentrations: Pharmaceutically acceptable salt of topotecan 12 mg / mL Cholesterol 12.14 mg / mL Phosphatidylcholine 95.93 mg / mL 1,2-Dioleoyl-3-trimethylpropylammonium chloride 24.31 mg / mL 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol 2.12 mg / mL.

5. The topotecan liposome according to any one of claims 1 to 4, characterized in that, The pharmaceutically acceptable salt of topotecan is topotecan hydrochloride.

6. A method for preparing topotecan liposomes according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) The cholesterol, phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol are dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve the pharmaceutically acceptable salt of the topotecan in buffer solution or sterile water for injection to obtain an aqueous phase; (3) Mix the lipid phase with the aqueous phase to obtain the final product.

7. The method for preparing topotecan liposomes according to claim 6, characterized in that, The organic solvent mentioned in step (1) is anhydrous ethanol; and / or, the buffer mentioned in step (2) is PBS buffer.

8. The method for preparing topotecan liposomes according to claim 6, characterized in that, In step (3), the lipid phase and the aqueous phase are mixed under the conditions of a total flow rate of 18 mL / min to 22 mL / min and a lipid phase to aqueous phase flow rate ratio of 0.8 to 1.2:

3.

9. The use of topotecan liposomes according to any one of claims 1 to 5 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is either metastatic ovarian cancer or small cell lung cancer.

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