Bortezomib encapsulating agent, bortezomib liposome and preparation method
By using negatively charged lipids and cholesterol to prepare bortezomib encapsulants and liposomes, the problems of complex preparation process, large particle size and low encapsulation rate in the prior art are solved, and small particle size and encapsulation are achieved. Preparation of bortezomib liposomes with high sealing rate.
Patent Information
- Application Number
- CN202411093236.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-08-11
- Filing Date
- 2024-08-09
- Publication Date
- 2025-02-25
AI Technical Summary
The existing bortezomib liposome preparation method has complex operation and large particle size. At the same time, the encapsulation rate of bortezomib is not high, making it difficult to meet the drug delivery needs of bortezomib.
Using negatively charged lipids and cholesterol as raw materials, bortezomib encapsulants and liposomes are prepared by lipid dispersion or solvent injection method. The specific steps include solution preparation, film formation dispersion and incubation, etc., and the preparation is carried out through these steps. Bortezomib liposomes with small particle size and high encapsulation rate are produced.
The efficient encapsulation of bortezomib liposomes is achieved, with a particle size controlled at about 100 nm and an encapsulation rate of more than 80%, simplifying the preparation process and reducing the operation complexity.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug carriers, and particularly relates to a bortezomib encapsulating agent, a bortezomib liposome and a preparation method thereof. Background Art
[0002] Bortezomib (BTZ) is a proteasome inhibitor, and its application in multiple myeloma (MM) has extended the median survival of MM from 3-5 years to 5-7 years. BTZ has become a Class I recommended drug for the treatment of newly diagnosed multiple myeloma (NDMM). However, adverse reactions such as peripheral neuropathy caused by BTZ seriously affect the survival and prognosis of patients. Since BTZ toxicity is caused by nonspecific interactions between BTZ and plasma proteins, it can be overcome by producing BTZ liposomes. Making liposomal BTZ is an effective way to reduce its drug side effects.
[0003] Liposomes (or lipid bilayer vesicles) are round vesicles composed of concentric ordered lipid bilayers that can encapsulate an aqueous phase, and can be used as delivery vehicles for therapeutic and diagnostic agents contained in an aqueous phase or lipid bilayer. Delivery of drugs in liposome encapsulated form can provide many advantages, depending on the drug, including, for example, reducing drug toxicity, changing pharmacokinetics, or increasing drug solubility. The existing preparation method of bortezomib liposomes is complicated, the particle size is large, and the encapsulation rate of bortezomib is not high. Therefore, a liposome with a simple preparation method is urgently needed to meet the administration of bortezomib. Summary of the invention
[0004] The object of the present invention is to provide a bortezomib encapsulating agent, a bortezomib liposome and a preparation method to solve at least one of the above problems.
[0005] According to one aspect of the present invention, a bortezomib encapsulating agent is provided. The encapsulating agent is a liposome, and its raw material components at least contain negatively charged lipids and cholesterol.
[0006] In some embodiments, the negatively charged lipid includes at least one of distearoylphosphatidylglycerol and its salts (DSPG), dipalmitoylphosphatidylglycerol and its salts (DPPG), dimyristoylphosphatidylglycerol and its salts (DMPG), dimyristoyl-sn-glycero-3-phosphate and its salts (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate and its salts (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphate and its salts (DOPA), 1,2-distearoyl-sn-glycero-3-phosphate and its salts (DSPA), dilauroyl phosphate and its salts (DLPA), and dipalmitoylphosphatidylserine and its salts (DPPS).
[0007] According to a second aspect of the present invention, a method for preparing a bortezomib encapsulating agent (i.e., blank liposome) is provided, wherein the encapsulating agent is a liposome, and the liposome is prepared by a lipid dispersion method, and the specific steps are as follows:
[0008] (1) adding purified water to meglumine until the concentration of the meglumine solution is 300 mM, and adding glacial acetic acid to adjust the pH value to 8.5 to obtain a meglumine buffer solution;
[0009] (2) dissolving cholesterol, negatively charged lipids and other raw material components in a first solvent, methanol and water composite solvent, and then dissolving in ethanol to obtain a lipid solution;
[0010] (3) evaporating the lipid solution in a water bath to remove the organic solvent;
[0011] (4) Adding a pH 8.5 meglumine solution to the solution after rotary evaporation in step (3) and hydrating in a 60° C. water bath, and then preparing blank liposomes by a 100 nm extrusion method.
[0012] In some embodiments, in step (2), the volume ratio of the first solvent, methanol, and water is 3:3:1.
[0013] In some embodiments, the first solvent in step (2) is at least one of chloroform, dichloromethane, ethanol, isopropyl acetate, isobutanol, and tert-butanol.
[0014] In some embodiments, the following step is further included: the blank liposomes prepared in step (4) are passed through a gel purification column and / or a hollow fiber column tangential flow ultrafiltration, and the meglumine in the external aqueous phase is removed using a NaCl-HEPES buffer having a pH of 6.5. Specifically, the gel purification column can be a G50 purification column.
[0015] The specific operation of preparing NaCl-HEPES buffer is as follows: weigh 1.6352g of sodium chloride, MW: 58.4; 0.9532g of HEPES, MW: 238.3, first add 150mL of pure water to dissolve completely, then add 1.0M sodium hydroxide solution to adjust the pH to 6.5, and then add pure water to 200mL. Among them, NaCl represents sodium chloride, HEPES represents 2-(4-(2-hydroxyethyl)piperazine)ethanesulfonic acid, CAS No. 7365-45-9; MW represents molecular weight.
[0016] Alternatively, the bortezomib encapsulating agent (i.e., blank liposome) can be obtained by solvent injection method, and the specific steps are as follows:
[0017] (1) preparing meglumine buffer solution with a pH of 8.5;
[0018] (2) dissolving cholesterol, negatively charged lipids and other raw material components with a second solvent, methanol and water composite solvent to obtain a lipid solution;
[0019] (3) The lipid solution was injected into the meglumine buffer at a volume ratio of 1:3, stirred at 60°C for 15 min, and then diluted with the meglumine buffer;
[0020] (4) removing the organic phase in the liposomes by tangential flow ultrafiltration using a gel purification column and / or a hollow fiber column and concentrating the liposome solution to 30 mg / ml, taking a sample and measuring the particle size;
[0021] (5) Blank liposomes were prepared using the 100 nm extrusion method.
[0022] In some embodiments, the second solvent is at least one of chloroform, dichloromethane, ethanol, isopropyl acetate, isobutanol, and tert-butanol.
[0023] In some embodiments, the volume ratio of the second solvent, methanol and water in step (2) is 2:8:1.5.
[0024] In some embodiments, the method further comprises the following steps: passing the blank liposomes prepared in step (5) through a gel purification column, and replacing the meglumine buffer in the external aqueous phase with a NaCl-HEPES buffer having a pH of 6.5 through a hollow fiber column tangential flow. The gel purification column may be a G50 purification column.
[0025] The specific operation of preparing NaCl-HEPES buffer is as follows: weigh 1.6352g of sodium chloride, MW: 58.4; 0.9532g of HEPES, MW: 238.3, first add 150mL of pure water to dissolve completely, then add 1.0M sodium hydroxide solution to adjust the pH to 6.5, and then add pure water to 200mL. Among them, NaCl represents sodium chloride, HEPES represents 2-(4-(2-hydroxyethyl)piperazine)ethanesulfonic acid, CAS No. 7365-45-9; MW represents molecular weight.
[0026] According to a third aspect of the present invention, a bortezomib liposome is provided, which is obtained by incubating bortezomib with the above-mentioned bortezomib encapsulating agent until bortezomib is loaded in the bortezomib encapsulating agent and removing free bortezomib.
[0027] According to a fourth aspect of the present invention, a method for preparing bortezomib liposomes is provided, and the specific steps are as follows:
[0028] (1) preparing blank liposomes, wherein the blank liposomes can be prepared by the above-mentioned lipid dispersion method or solvent injection method;
[0029] (2) Add bortezomib ethanol solution to blank liposomes and incubate at room temperature;
[0030] (3) Free bortezomib is removed to obtain a liposome solution containing bortezomib.
[0031] In some embodiments, the preparation steps of the bortezomib ethanol solution in step (2) are as follows: 3.84 mg of bortezomib is dissolved in 200 μl of anhydrous ethanol.
[0032] In some embodiments, in step (3), free bortezomib can be removed by passing through a G50 purification column, or by tangential flow through a hollow fiber column.
[0033] The bortezomib liposome encapsulation rate prepared by the invention reaches more than 80%, and the particle size is relatively small, reaching about 100 nm. DETAILED DESCRIPTION
[0034] The present invention will be further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the following raw materials are commercially available.
[0035] Example 1
[0036] Example 1 Bortezomib liposomes were prepared by lipid dispersion method, and the specific steps were as follows:
[0037] 1. Solution preparation
[0038] (1) Preparation of meglumine buffer: 488 mg (2.5 mmol) of meglumine (molecular weight 195.2) was added to 8.33 ml of pure water. At this point, the concentration of the meglumine solution was 300 mM. Glacial acetic acid was added to adjust the pH to 8.5 to obtain meglumine buffer for later use.
[0039] (2) Preparation of NaCl-HEPES buffer at pH 6.5: Prepare 200 mL of 140 mM NaCl and 20 mM HEPES buffer. Weigh 1.6352 g of sodium chloride (MW: 58.4) and 0.9532 g of HEPES (MW: 238.3). First, add 150 mL of pure water to dissolve completely. Then, add 1.0 M sodium hydroxide solution to adjust the pH to 6.5 and then add pure water to 200 mL.
[0040] (3) Preparation of lipid solution: Cholesterol, DSPC, and DSPG were weighed according to the formula in Table 1, and 1.4 ml of a composite solvent of chloroform, methanol, and water (volume ratio of 0.6:0.6:0.2) was added, and then 4 ml of ethanol was added to dissolve the lipid solution.
[0041] Table 1 Formula of each component
[0042]
[0043] 2. Preparation of Bortezomib Liposomes by Film Formation and Dispersion
[0044] The lipid solution obtained by the above operation is subjected to the following operations:
[0045] (1) Rotary evaporation at 45°C in a water bath to remove the organic solvent;
[0046] (2) First add 5 ml of meglumine solution with a pH of 8.5 and hydrate in a 60°C water bath;
[0047] (3) Preparation of liposomes using the 100 nm extrusion method;
[0048] (4) passing through a G50 purification column and using a NaCl-HEPES buffer at pH 6.5 to remove meglumine from the external aqueous phase;
[0049] (5) Add bortezomib ethanol solution (3.84 mg bortezomib dissolved in 200 μl anhydrous ethanol) and incubate at room temperature for 12 h;
[0050] (6) The free bortezomib was removed by passing through a G50 purification column to obtain a liposome solution loaded with bortezomib.
[0051] Example 2
[0052] Bortezomib liposomes were prepared by solvent injection method.
[0053] 1. Solution preparation
[0054] (1) Preparation of meglumine buffer: 488 mg (2.5 mmol) of meglumine (molecular weight 195.2) was added to 8.33 ml of pure water. At this point, the concentration of the meglumine solution was 300 mM. Glacial acetic acid was added to adjust the pH to 8.5 to obtain meglumine buffer for later use.
[0055] (2) Preparation of NaCl-HEPES buffer at pH 6.5: Prepare 200 mL of 140 mM NaCl and 20 mM HEPES buffer. Weigh 1.6352 g of sodium chloride (MW: 58.4) and 0.9532 g of HEPES (MW: 238.3). First, add 150 mL of pure water to dissolve completely. Then, add 1.0 M sodium hydroxide solution to adjust the pH to 6.5 and then add pure water to 200 mL.
[0056] (3) Preparation of lipid solution: Cholesterol, DSPC, and DSPG were weighed according to the formula in Table 2, and 10 mL of a composite solvent of isopropyl acetate, ethanol, and water (volume ratio of 2:8:1.5) was used to dissolve the lipids to obtain a lipid solution.
[0057] Table 2 Formula of each component
[0058]
[0059] 2. Preparation of Bortezomib Liposomes by Solvent Injection
[0060] (1) Inject the organic solvent dissolving lipids into the meglumine buffer according to the conditions in Table 3, stir at 60°C for 15 min, and then dilute with 20 mL of meglumine buffer.
[0061] Table 3 Condition parameters
[0062] Water bath temperature Solvent ratio (lipid organic phase: meglumine buffer) 60℃ 5mL injected into 15mL (1:3)
[0063] (2) removing the organic phase from the liposomes by tangential flow through a hollow fiber column and concentrating the liposome solution to 30 mg / ml, and taking samples to measure the particle size;
[0064] (3) Preparation of liposomes using the 100 nm extrusion method;
[0065] (4) replacing the meglumine buffer in the external aqueous phase with NaCl-HEPES buffer at pH 6.5 by tangential flow through the hollow fiber column;
[0066] (5) Add bortezomib ethanol solution (3.84 mg dissolved in 200 μl ethanol), incubate at room temperature for different time periods, and measure the total concentration of bortezomib liposomes;
[0067] (6) The free bortezomib is removed by tangential flow in the hollow fiber column to obtain a liposome solution loaded with bortezomib.
[0068] Example 3
[0069] Bortezomib liposomes were prepared by solvent injection method.
[0070] 1. Solution preparation
[0071] (1) Preparation of meglumine buffer: 488 mg (2.5 mmol) of meglumine (molecular weight 195.2) was added to 8.33 ml of pure water. At this point, the concentration of the meglumine solution was 300 mM. Glacial acetic acid was added to adjust the pH to 8.5 to obtain meglumine buffer for later use.
[0072] (2) Preparation of NaCl-HEPES buffer at pH 6.5: Prepare 200 mL of 140 mM NaCl and 20 mM HEPES buffer. Weigh 1.6352 g of sodium chloride (MW: 58.4) and 0.9532 g of HEPES (MW: 238.3). First, add 150 mL of pure water to dissolve completely. Then, add 1.0 M sodium hydroxide solution to adjust the pH to 6.5 and then add pure water to 200 mL.
[0073] (3) Preparation of lipid solution: Cholesterol, DSPC, DSPG, and DSPE-PEG2K were weighed according to the formula in Table 4, and 10 mL of a composite solvent of isopropyl acetate, ethanol, and water (volume ratio of 2:8:1.5) was used to dissolve the lipids to obtain a lipid solution.
[0074] Table 4 Formula of each component
[0075]
[0076] 2. Preparation of Bortezomib Liposomes by Solvent Injection
[0077] (1) Inject the organic solvent dissolving lipids into the meglumine buffer according to the conditions in Table 5, stir at 60°C for 15 min, and then dilute with 20 mL of meglumine buffer.
[0078] Table 5 Condition parameters
[0079] Water bath temperature Solvent ratio (lipid organic phase: meglumine buffer) 60℃ 5mL injected into 15mL (1:3)
[0080] (2) removing the organic phase from the liposomes by tangential flow through a hollow fiber column and concentrating the liposome solution to 30 mg / ml, and taking samples to measure the particle size;
[0081] (3) Preparation of liposomes using the 100 nm extrusion method;
[0082] (4) replacing the meglumine buffer in the external aqueous phase with NaCl-HEPES buffer at pH 6.5 by tangential flow through the hollow fiber column;
[0083] (5) Add bortezomib ethanol solution (3.84 mg dissolved in 200 μl ethanol), incubate at room temperature for different time periods, and measure the total concentration of bortezomib liposomes;
[0084] (6) The free bortezomib is removed by tangential flow in the hollow fiber column to obtain a liposome solution loaded with bortezomib.
[0085] Example 4
[0086] Bortezomib liposomes were prepared by solvent injection method.
[0087] 1. Solution preparation
[0088] (1) Preparation of meglumine buffer: 488 mg (2.5 mmol) of meglumine (molecular weight 195.2) was added to 8.33 ml of pure water. At this point, the concentration of the meglumine solution was 300 mM. Glacial acetic acid was added to adjust the pH to 8.5 to obtain meglumine buffer for later use.
[0089] (2) Preparation of NaCl-HEPES buffer at pH 6.5: Prepare 200 mL of 140 mM NaCl and 20 mM HEPES buffer. Weigh 1.6352 g of sodium chloride (MW: 58.4) and 0.9532 g of HEPES (MW: 238.3). First, add 150 mL of pure water to dissolve completely. Then, add 1.0 M sodium hydroxide solution to adjust the pH to 6.5 and then add pure water to 200 mL.
[0090] (3) Preparation of lipid solution: Cholesterol, DSPC, DSPG, and cholesterol-PEG2K were weighed according to the formula in Table 6, and 10 mL of a composite solvent of isopropyl acetate, ethanol, and water (volume ratio of 2:8:1.5) was used to dissolve the lipids to obtain a lipid solution.
[0091] Table 6 Formula of each component
[0092]
[0093] 2. Preparation of Bortezomib Liposomes by Solvent Injection
[0094] (1) Inject the organic solvent dissolving lipids into the meglumine buffer according to the conditions in Table 7, stir at 60°C for 15 min, and then dilute with 20 mL of meglumine buffer.
[0095] Table 7 Condition parameters
[0096] Water bath temperature Solvent ratio (lipid organic phase: meglumine buffer) 60℃ 5mL injected into 15mL (1:3)
[0097] (2) removing the organic phase from the liposomes through tangential flow through a hollow fiber column and concentrating the liposome solution to 30 mg / ml, then taking a sample to measure the particle size;
[0098] (3) Preparation of liposomes using the 100 nm extrusion method;
[0099] (4) replacing the meglumine buffer in the external aqueous phase with NaCl-HEPES buffer at pH 6.5 by tangential flow through the hollow fiber column;
[0100] (5) Add bortezomib ethanol solution (3.84 mg dissolved in 200 μl ethanol), incubate at room temperature for different time periods, and measure the total concentration of bortezomib liposomes;
[0101] (6) The free bortezomib is removed by tangential flow in the hollow fiber column to obtain a liposome solution loaded with bortezomib.
[0102] The liposomes prepared according to Examples 1, 2, 3 and 4 were measured using a Malvern laser particle size analyzer, and the encapsulation efficiency was measured using a high performance liquid chromatography. The results are shown in Table 8 below.
[0103] Table 8 Experimental results
[0104] Preparation method Encapsulation efficiency Particle size PDI Embodiment 1 80.1％ 108nm 0.03 Embodiment 2 82.1％ 106.9nm 0.03 Embodiment 3 96.3％ 104nm 0.09 Embodiment 4 77.4％ 102.8nm 0.07
[0105] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. Bortezomib encapsulating agent, characterized in that, The encapsulating agent is a liposome, and its raw material components at least contain negatively charged lipids and cholesterol.
2. The bortezomib encapsulating agent according to claim 1, characterized in that: The negatively charged lipids include at least one of distearoylphosphatidylglycerol and its salts, dipalmitoylphosphatidylglycerol and its salts, dimyristoylphosphatidylglycerol and its salts, dimyristoyl-sn-glycero-3-phosphate and its salts, 1,2-dipalmitoyl-sn-glycero-3-phosphate and its salts, 1,2-dioleoyl-sn-glycero-3-phosphate and its salts, 1,2-distearoyl-sn-glycero-3-phosphate and its salts, dilauroyl phosphate and its salts, and dipalmitoylphosphatidylserine and its salts.
3. The method for preparing the bortezomib encapsulating agent according to claim 1, characterized in that: The lipid dispersion method was used for preparation, and the specific steps were as follows: (1) preparing meglumine buffer; (2) dissolving the raw material components of the bortezomib encapsulating agent according to claim 1 in a first solvent, methanol, and a water composite solvent, and then dissolving in ethanol to obtain a lipid solution; (3) subjecting the lipid solution described in step (2) to rotary evaporation; (4) adding the meglumine solution to the lipid solution after rotary evaporation in step (3), and then preparing the bortezomib encapsulating agent by a 100 nm extrusion method.
4. The preparation method according to claim 3, characterized in that: In step (2), the first solvent is at least one of chloroform, dichloromethane, ethanol, isopropyl acetate, isobutyl alcohol and tert-butyl alcohol.
5. The preparation method according to claim 3 or 4, characterized in that: The method further comprises the following steps: preparing NaCl-HEPES buffer, passing the blank liposomes prepared in step (4) through a gel purification column and / or a hollow fiber column tangential flow ultrafiltration, and removing meglumine in the external aqueous phase with NaCl-HEPES buffer.
6. The method for preparing the bortezomib encapsulating agent according to claim 1, characterized in that: The solvent injection method is used for preparation, and the specific steps are as follows: (1) preparing meglumine buffer; (2) dissolving the raw material components of the bortezomib encapsulating agent according to claim 1 in a second solvent / methanol / water composite solvent to obtain a lipid solution; (3) injecting the lipid solution into the meglumine buffer, stirring and then diluting with the meglumine buffer; (4) removing the organic phase in the liposomes by tangential flow ultrafiltration using a gel purification column and / or a hollow fiber column, concentrating the liposome solution, and taking samples to measure the particle size; (5) Liposomes were prepared using the 100 nm extrusion method.
7. The preparation method according to claim 6, characterized in that: In step (2), the second solvent is at least one of chloroform, dichloromethane, ethanol, isopropyl acetate, isobutyl alcohol and tert-butyl alcohol.
8. A bortezomib liposome, characterized in that: Bortezomib is incubated with the bortezomib encapsulating agent according to claim 1 until bortezomib is loaded in the bortezomib encapsulating agent, and free bortezomib is removed.
9. The method for preparing the bortezomib liposome according to claim 8, characterized in that: Here are the steps: (1) preparing a bortezomib encapsulating agent; (2) adding bortezomib ethanol solution to the bortezomib encapsulating agent and incubating at room temperature; (3) removing free bortezomib to obtain bortezomib liposomes.
10. The preparation method according to claim 9, characterized in that: In step (3), free bortezomib can be removed by a gel purification column, or by tangential flow of a hollow fiber column.