Liposomal pemetrexed and methods of making and using the same

Abstract

The application discloses a kind of pemetrexed liposome and its preparation method and application, it is made of including 2mg / mL~20mg / mL pemetrexed or its pharmaceutically acceptable salt, 2.2mg / mL~12mg / mL cholesterols, 6.81mg / mL~108mg / mL neutral lipid, 12mg / mL~98mg / mL cationic lipid and 0.12mg / mL~2.12mg / mL PEG-lipid.The pemetrexed liposome of the application can significantly improve the enrichment of pemetrexed drug in tumor site, thereby enhancing drug treatment effect, reducing dosing dose, reducing the toxic side effects of drug on normal tissue.And it has good stability, no obvious change in storage process nature, particle size, zeta potential and encapsulation efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a pemetrexed liposome, its preparation method, and its application. Background Technology

[0002] Pemetrexed is a multi-target anticancer folate antagonist that inhibits cell replication by disrupting key folate-dependent metabolic processes essential for cell replication. In vitro studies show that pemetrexed exerts its effects by inhibiting the activity of thymidine synthase (TS), dihydrofolate reductase (DHFR), and glycine nucleotide formyltransferase (GARFT), all of which are key folate-dependent enzymes in the biosynthesis of thymidine nucleotides and purine nucleotides. Pemetrexed effectively inhibits the growth of various tumor cell lines, including mesothelioma and lung cancer. When used in combination with drugs such as cisplatin, it produces a synergistic therapeutic effect. It is currently widely used in the clinical treatment of malignant pleural mesothelioma, non-small cell lung cancer, and other malignant tumors, and is one of the core drugs in first-line chemotherapy regimens.

[0003] Developed by Lilly, pemetrexed powder for injection was first launched in the United States on February 4, 2004, and is now available in China. This powder for injection is used to treat inoperable malignant pleural mesothelioma and non-squamous non-small cell lung cancer. Currently, no other formulations are available. Because pemetrexed powder for injection lacks tumor targeting and is widely distributed in normal tissues and organs, its efficacy is not significant. Furthermore, it produces non-specific cytotoxicity to rapidly proliferating normal cells such as bone marrow cells, gastrointestinal cells, and reproductive system cells, easily causing serious adverse reactions such as bone marrow suppression, gastrointestinal reactions, and reproductive toxicity, reducing patient tolerance and treatment adherence.

[0004] There are also reports on pemetrexed disodium liposomes, most of which encapsulate pemetrexed in neutral lipids, which reduces the side effects of pemetrexed to some extent and improves efficacy. However, pemetrexed disodium liposomes lack targeting and cannot effectively target and enrich tumor sites. Summary of the Invention

[0005] The purpose of this invention is to provide a pemetrexed liposome with strong tumor targeting.

[0006] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0007] A first aspect of the present invention provides a pemetrexed liposome, which is made from raw materials comprising the following concentrations: Pemetrexed or its pharmaceutically acceptable salt 2 mg / mL to 20 mg / mL Cholesterol 2.2 mg / mL ~ 12 mg / mL Neutral lipids: 6.81 mg / mL ~ 108 mg / mL Cationic lipids 12 mg / mL ~ 98 mg / mL PEG-lipids 0.12 mg / mL ~ 2.12 mg / mL; The cationic lipid is one or more of 1,2-bisoctadecenoxy-3-methylammonium propane chloride, dioleoyl-2,3-diaminopropylammonium, (2,3-dioleoyl-propyl)-trimethylamine (chloride), and 1,2-dilinoleoyloxy-3-dimethylaminopropane.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned pemetrexed liposomes, comprising the following steps: (1) Cholesterol, neutral lipids, cationic lipids and PEG-lipids were dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve pemetrexed or a pharmaceutically acceptable salt thereof in a buffer solution to obtain an aqueous phase; (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 to aqueous phase flow rate ratio of 0.8 to 1.2:3 to obtain the final product.

[0009] In a third aspect, the present invention provides the use of the above-described pemetrexed liposomes in the preparation of a medicament for treating lung cancer.

[0010] The inventors of this invention have discovered that by using a lipid phase comprising cationic lipids as a carrier to encapsulate pemetrexed or a pharmaceutically acceptable salt thereof, pemetrexed liposomes can be prepared. When these liposomes are administered to tumor-bearing mice, they can significantly increase the accumulation of pemetrexed in the lungs, thereby enhancing the therapeutic effect, reducing the dosage, and decreasing the toxic side effects of the drug on normal tissues.

[0011] The pemetrexed liposomes of the present invention have good stability, and their properties, particle size, zeta potential and encapsulation efficiency do not change significantly during storage. Attached Figure Description

[0012] Figure 1 This is a comparison chart of the accumulation of samples 13, 14, and 15 in the lungs at 1h, 6h, and 24h after administration of the drug in this invention.

[0013] Figure 2 The images show in vivo lung imaging of samples 13, 14, and 15 in mice 1 hour after administration of the drug in this invention. The fourth image from the left is the lung imaging result.

[0014] Figure 3 The images show in vivo lung imaging of samples 13, 14, and 15 in mice 6 hours after drug administration in this invention. The fourth image from the left is the lung imaging result.

[0015] Figure 4 The images show in vivo lung imaging of samples 13, 14, and 15 in mice 24 hours after drug administration in this invention. The fourth image from the left is the lung imaging result. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more complete 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.

[0017] 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.

[0018] 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.

[0019] In some embodiments of the present invention, a pemetrexed liposome is disclosed, which is made from raw materials comprising the following concentrations: Pemetrexed or its pharmaceutically acceptable salt 2 mg / mL to 20 mg / mL Cholesterol 2.2 mg / mL ~ 12 mg / mL Neutral lipids: 6.81 mg / mL ~ 108 mg / mL Cationic lipids 12 mg / mL ~ 98 mg / mL PEG-lipids 0.12 mg / mL ~ 2.12 mg / mL; The cationic lipid is one or more of 1,2-bisoctadecenoxy-3-methylammonium propane chloride (DOTMA), dioleoyl-2,3-diaminopropylammonium (DODAP), (2,3-dioleoyl-propyl)-trimethylamine (chloride) (DOTAP), and 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLin-MC3-DMA).

[0020] In this invention, a lipid phase comprising cholesterol, neutral lipids, cationic lipids, and PEG-lipids is used as a carrier to encapsulate pemetrexed or its pharmaceutically acceptable salt, thereby preparing pemetrexed liposomes. Because the surface of pulmonary vascular endothelial cells is covered by a thicker glycocalyx layer than in other parts of the body, rich in negatively charged glycosaminoglycans such as heparan sulfate (HS), when pemetrexed liposomes are administered, the positive charge on the surface of the cationic lipids in the pemetrexed liposomes forms a strong electrostatic attraction with the negative charge of the glycocalyx layer, allowing the liposomes to rapidly and specifically adsorb onto the surface of the pulmonary vascular endothelium. This enables targeting of the lungs and increases the accumulation of the drug within lung cancer cells. Therefore, the pemetrexed liposomes of this invention show great promise in the treatment of lung cancer.

[0021] In one embodiment, the cationic lipid is (2,3-dioleoyl-propyl)-trimethylamine (chloride).

[0022] In one embodiment, the concentration ratio of the cationic lipid to the neutral lipid is 0.1 to 6.2:1.

[0023] In one embodiment, the concentration ratio of the cationic lipid to the neutral lipid is 0.25 to 6:1.

[0024] In one embodiment, the concentration ratio of the cationic lipid to the neutral lipid is 1 to 6:1.

[0025] In one embodiment, the concentration ratio of the cationic lipid to the neutral lipid is 3 to 6:1.

[0026] In one embodiment, the concentration ratio of the cationic lipid to the neutral lipid is 5-6:1.

[0027] In one embodiment, the neutral lipid is one or more of phosphatidylcholine (PC), hydrogenated soybean phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), and myristoylphosphatidylcholine (DMPC).

[0028] In one embodiment, the neutral lipid is phosphatidylcholine (PC) or distearate phosphatidylcholine (DSPC).

[0029] In one embodiment, the PEG-lipid is one or more of distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), distearylphosphatidylethanolamine-polyethylene glycol 2000-azide (DSPE-PEG2000-N3), and distearylphosphatidylethanolamine-polyethylene glycol 2000-amino (DSPE-PEG2000-NH2).

[0030] In one embodiment, the PEG-lipid is distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000).

[0031] In one embodiment, the pemetrexed or a pharmaceutically acceptable salt thereof is pemetrexed disodium.

[0032] In one embodiment, the pemetrexed liposome is made from raw materials comprising the following concentrations: Pemetrexed disodium 2 mg / mL ~ 4 mg / mL Cholesterol 2.2 mg / mL ~ 3.2 mg / mL Neutral lipids 6.81 mg / mL ~ 7 mg / mL Cationic lipids 12 mg / mL~15 mg / mL PEG-lipids 0.12 mg / mL ~ 0.2 mg / mL.

[0033] In one embodiment, the pemetrexed liposome is made from raw materials comprising the following concentrations: Pemetrexed disodium 2mg / mL~10mg / mL Cholesterol 10mg / mL~12mg / mL Neutral lipids 100 mg / mL ~ 108 mg / mL Cationic lipids 12 mg / mL~15 mg / mL PEG-lipids 2 mg / mL ~ 2.12 mg / mL.

[0034] In other embodiments of the present invention, a method for preparing the above-mentioned pemetrexed liposomes is disclosed, comprising the following steps: (1) Cholesterol, neutral lipids, cationic lipids and PEG-lipids were dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve pemetrexed or a pharmaceutically acceptable salt thereof in a buffer solution to obtain an aqueous phase; (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 to aqueous phase flow rate ratio of 0.8 to 1.2:3 to obtain the final product.

[0035] In one embodiment, the organic solvent in step (1) is anhydrous ethanol.

[0036] In one embodiment, the buffer solution in step (2) is a PBS buffer solution.

[0037] In other embodiments of the present invention, the use of the above-mentioned pemetrexed liposomes in the preparation of drugs for treating malignant pleural mesothelioma or non-small cell lung cancer is disclosed.

[0038] All the raw materials used in the following examples are commercially available.

[0039] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] Example 1: Pemetrexed liposomes and their preparation method In this embodiment, pemetrexed disodium liposomes (sample 1) were prepared, and their formulation is shown in Table 1.

[0041]

[0042] The preparation method includes the following steps: (1) Dissolve pemetrexed disodium in PBS buffer to obtain the aqueous phase.

[0043] (2) Distearate phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000, and (2,3-dioleoyl-propyl)-trimethylamine (chloride salt) were dissolved in anhydrous ethanol to obtain the lipid phase.

[0044] (3) Using a microfluidic device, the above lipid phase and water phase are rapidly mixed at a total flow rate of 20 mL / min and a lipid (lipid phase) to water (water phase) flow rate ratio of 1:3 to obtain the final product.

[0045] The properties, particle size, zeta potential, and drug loading of sample 1 were tested. The results are shown in Table 2.

[0046]

[0047] As shown in Table 2, the pemetrexed liposomes prepared in Example 1 exhibited uniform properties, without stratification or aggregation, with an average particle size of 252.7 nm, which is relatively small. The zeta potential was 16.9 mV, indicating a positive potential, and the drug loading was 39.3%. After being stored at 2–8 °C for 30 days, there were no significant changes in its properties, particle size, zeta potential, or drug loading.

[0048] Example 2: Pemetrexed liposomes and their preparation method In this embodiment, samples 2 to 5 were prepared according to the method of Example 1, and their formulations are shown in Table 3.

[0049]

[0050] The properties, particle size, and encapsulation efficiency of samples 2 through 5 were tested. The results are shown in Table 4.

[0051]

[0052] Table 4 shows that when the concentration of pemetrexed in liposomes varies within a certain range, there are no significant differences in its properties, particle size, and encapsulation efficiency.

[0053] Example 3: Pemetrexed liposomes and their preparation method In this embodiment, samples 6 and 7 were prepared according to the method of Example 1, and their formulations are shown in Table 5.

[0054]

[0055] The properties, particle size, zeta potential, and encapsulation efficiency of samples 6 and 7 were tested. The results are shown in Table 6.

[0056]

[0057] The results in Table 6 show that there are little difference in the properties, particle size, zeta potential and encapsulation efficiency between samples 6 and 7.

[0058] Example 4: Pemetrexed liposomes and their preparation method In this embodiment, samples 8 to 10 were prepared according to the method of Example 1, and their formulations are shown in Table 7.

[0059]

[0060] The properties, particle size, zeta potential, and encapsulation efficiency of samples 8-10 were measured. The results are shown in Table 8.

[0061]

[0062] Table 8 shows that adjusting the ratio of neutral lipids to cationic lipids has little effect on properties and particle size, but it has a significant effect on zeta potential and encapsulation efficiency. The higher the proportion of cationic lipids, the higher the zeta potential and encapsulation efficiency.

[0063] Example 5: Pemetrexed liposomes and their preparation method In this embodiment, samples 11 and 12 were prepared according to the method of Example 1, and their formulations are shown in Table 9.

[0064]

[0065] The properties, particle size, and zeta potential of samples 11 and 12 were tested. The results are shown in Table 10.

[0066]

[0067] The results in Table 10 show that adjusting the ratio of neutral lipids to cationic lipids has little effect on properties and particle size, but it has a significant effect on zeta potential. The higher the proportion of cationic lipids, the higher the zeta potential.

[0068] Example 6: Comparison of the effects of different pemetrexed liposomes on tumor targeting This example compares the effects of different pemetrexed liposomes (formulations shown in Table 11) on tumor targeting.

[0069]

[0070] The experimental method is as follows: Models were established using C57BL / 6 mice and B16F10 tumor cells. Mice bearing B16F10 tumors were intravenously administered samples 13 (cationic lipid DOTAP), 14 (cationic lipid MC3), and 15 (excluding cationic lipids) at a dose of 12 mg / kg. In vivo imaging was performed at 1 h, 6 h, and 24 h post-administration to examine the accumulation of the three liposomes in the lungs over time.

[0071] The results are shown in Table 12 and Figures 1-4 As shown.

[0072]

[0073] From Table 12 and Figures 1-4 The results showed that at 1 h after administration, the cumulative amount of sample 13 in the lungs was significantly higher than that of samples 14 and 15. At 6 h and 24 h after administration, the cumulative amounts of samples 13 and 14 in the lungs were both higher than that of sample 15 to some extent.

[0074] The results of this study indicate that pemetrexed liposomes prepared from cationic lipids, especially DOTAP, can provide a strong ability to target the lungs, further enriching the drug at the tumor site, thereby enhancing the therapeutic effect, reducing the dosage, and reducing the toxic side effects of the drug on normal tissues.

[0075] 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.

[0076] 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 pemetrexed liposome, characterized in that, It is made from raw materials containing the following concentrations: Pemetrexed or its pharmaceutically acceptable salt 2 mg / mL to 20 mg / mL Cholesterol 2.2 mg / mL ~ 12 mg / mL Neutral lipids: 6.81 mg / mL ~ 108 mg / mL Cationic lipids 12 mg / mL ~ 98 mg / mL PEG-lipids 0.12 mg / mL ~ 2.12 mg / mL; The cationic lipid is one or more of 1,2-bisoctadecenoxy-3-methylammonium propane chloride, dioleoyl-2,3-diaminopropylammonium, (2,3-dioleoyl-propyl)-trimethylamine (chloride), and 1,2-dilinoleoyloxy-3-dimethylaminopropane, preferably (2,3-dioleoyl-propyl)-trimethylamine (chloride).

2. The pemetrexed liposome according to claim 1, characterized in that, The concentration ratio of the cationic lipid to the neutral lipid is 0.1~6.2:1, preferably 0.25~6:1, more preferably 1~6:1, and even more preferably 5~6:

1.

3. The pemetrexed liposome according to claim 1, characterized in that, The neutral lipid is one or more of phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, and dimyristoyl phosphatidylcholine.

4. The pemetrexed liposome according to claim 1, characterized in that, The PEG-lipid is one or more of the following: distearylphosphatidylethanolamine-polyethylene glycol 2000, distearylphosphatidylethanolamine-azide polyethylene glycol 2000, and distearylphosphatidylethanolamine-polyethylene glycol-amino.

5. The pemetrexed liposome according to claim 1, characterized in that, The pemetrexed or its pharmaceutically acceptable salt is pemetrexed disodium.

6. The pemetrexed liposome according to any one of claims 1 to 5, characterized in that, Made from raw materials containing the following concentrations: Pemetrexed disodium 2 mg / mL ~ 4 mg / mL Cholesterol 2.2 mg / mL ~ 3.2 mg / mL Neutral lipids 6.81 mg / mL ~ 7 mg / mL Cationic lipids 12 mg / mL~15 mg / mL PEG-lipids 0.12 mg / mL ~ 0.2 mg / mL.

7. The pemetrexed liposome according to any one of claims 1 to 5, characterized in that, Made from raw materials containing the following concentrations: Pemetrexed disodium 2mg / mL~10mg / mL Cholesterol 10mg / mL~12mg / mL Neutral lipids 100 mg / mL ~ 108 mg / mL Cationic lipids 12 mg / mL~15 mg / mL PEG-lipids 2 mg / mL ~ 2.12 mg / mL.

8. The method for preparing pemetrexed liposomes according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Cholesterol, neutral lipids, cationic lipids and PEG-lipids were dissolved in an organic solvent to obtain the lipid phase; (2) Dissolve pemetrexed or a pharmaceutically acceptable salt thereof in a buffer solution to obtain an aqueous phase; (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 to aqueous phase flow rate ratio of 0.8 to 1.2:3 to obtain the final product.

9. The method for preparing pemetrexed liposomes according to claim 8, characterized in that, The organic solvent mentioned in step (1) is anhydrous ethanol, and the buffer solution mentioned in step (2) is PBS buffer.

10. The use of the pemetrexed liposome according to any one of claims 1 to 7 in the preparation of a medicament for treating lung cancer.

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