A tolfenamic acid liposome, a preparation method and application thereof

Abstract

This invention relates to a tofenamic acid liposome, its preparation method, and its application. The raw materials for preparing the tofenamic acid liposome include phospholipids, cholesterol, and tofenamic acid. The tofenamic acid is encapsulated in a lipid bilayer. The tofenamic acid liposome of this invention improves the solubility of tofenamic acid and has a high encapsulation efficiency and drug loading capacity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, and relates to a tofenamic acid liposome, its preparation method and application. Background Technology

[0002] Tofenamic acid is a member of the anthranilic acid derivative class of NSAID drugs discovered by scientists at the Finnish pharmaceutical company Medica. Clinically, it primarily functions as an anti-inflammatory, rapid antipyretic, and analgesic. Tofenamic acid has been approved for both human and veterinary use. Extensive research data indicates that tofenamic acid is rapidly absorbed in animals, reaching peak absorption within 1-2 hours after injection, and has not shown serious specific toxic reactions, thus establishing it as a relatively safe veterinary analgesic.

[0003] However, tofenamic acid has extremely low solubility in water, making it unsuitable for use as an injectable solution, which causes inconvenience during administration and affects the therapeutic effect. Therefore, there is an urgent need to provide a drug that can improve the solubility of tofenamic acid while ensuring safety. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a tofenamic acid liposome, its preparation method, and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a tofenamic acid liposome, wherein the raw materials for preparing the liposome include phospholipids, cholesterol and tofenamic acid, and the tofenamic acid is encapsulated in a phospholipid bilayer.

[0007] The tofenamic acid liposomes described in this invention improve the solubility of tofenamic acid and have a high encapsulation efficiency and drug loading.

[0008] Preferably, the raw materials for preparing the liposomes include 10-12 parts phospholipids, 2-6 parts cholesterol, and 1-1.5 parts tofenamic acid by mass.

[0009] The mass fractions of the phospholipids can be selected from 10 parts, 10.1 parts, 10.2 parts, 10.3 parts, 10.4 parts, 10.5 parts, 10.6 parts, 10.7 parts, 10.8 parts, 10.9 parts, 11 parts, 11.1 parts, 11.2 parts, 11.3 parts, 11.4 parts, 11.5 parts, 11.6 parts, 11.7 parts, 11.8 parts, 11.9 parts, 12 parts, etc., and the mass fractions of the cholesterol can be selected from 2 parts, 2.2 parts, 2. The quantities of tofenamic acid can be 4 parts, 2.6 parts, 2.8 parts, 3 parts, 3.2 parts, 3.4 parts, 3.6 parts, 3.8 parts, 4 parts, 4.2 parts, 4.4 parts, 4.6 parts, 4.8 parts, 5 parts, 5.2 parts, 5.4 parts, 5.6 parts, 5.8 parts, 6 parts, etc. The mass fractions of tofenamic acid can be selected as 1 part, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0010] When added in the above-mentioned proportions by weight, the encapsulation efficiency and drug loading of liposomes are better, resulting in better efficacy.

[0011] Preferably, the raw materials for preparing the liposomes further include a solubilizer.

[0012] The addition of solubilizers helps to stabilize the dispersion of liposomes and increases their stability.

[0013] Preferably, the solubilizer comprises DSPE-PEG2000.

[0014] Preferably, the phospholipid comprises any one or a combination of at least two of the following: hydrogenated phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearate phosphatidylcholine, dioleoyl phosphatidylcholine, 1,2-tetradecanoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, or lecithin.

[0015] Preferably, the phospholipid is hydrogenated phosphatidylcholine.

[0016] This invention creatively discovers that hydrogenated phosphatidylcholine is more effective than other phospholipids.

[0017] Preferably, the pH of the liposomes is 6-7, such as 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0018] In a second aspect, the present invention provides a method for preparing tofenamic acid liposomes according to the first aspect, the method comprising:

[0019] (1) Mix phospholipids, cholesterol, tofenamic acid with an organic solvent and rotary evaporate until a thin film forms on the container wall;

[0020] (2) Add deionized water to the container in step (1) and sonicate to obtain a dispersion;

[0021] (3) The dispersion obtained in step (2) is homogenized under high pressure to obtain the final product.

[0022] Preferably, step (2) further includes mixing the dispersion with a pH adjuster.

[0023] Preferably, the pH adjuster comprises disodium succinate hexahydrate.

[0024] Preferably, the organic solvent includes methanol and / or chloroform.

[0025] Preferably, the rotary evaporation temperature is 36-38℃ and the rotation speed is 90-110 rpm.

[0026] The temperature can be selected from 36℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃, 38℃, etc., and the rotation speed can be selected from 90rpm, 92rpm, 94rpm, 96rpm, 98rpm, 100rpm, 102rpm, 104rpm, 106rpm, 108rpm, 110rpm, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0027] Preferably, the addition of deionized water further includes a 60-65°C water bath for 15-25 minutes.

[0028] The water bath temperature can be selected from 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, etc., and the water bath time can be selected from 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0029] Preferably, the ultrasonic power is 400-600W and the duration is 10-15min.

[0030] The ultrasonic power can be selected from 400W, 420W, 440W, 460W, 480W, 500W, 520W, 540W, 560W, 580W, 600W, etc., and the time can be selected from 10min, 11min, 12min, 13min, 14min, 15min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0031] Preferably, the high-pressure homogenization is performed at a pressure of 15,000-20,000 psi for 3-5 seconds.

[0032] The pressure for high-pressure homogenization can be selected from 15000psi, 15500psi, 16000psi, 16500psi, 17000psi, 17500psi, 18000psi, 18500psi, 19000psi, 19500psi, 20000psi, etc., and the time can be selected from 3s, 3.2s, 3.4s, 3.6s, 3.8s, 4s, 4.2s, 4.4s, 4.6s, 4.8s, 5s, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0033] Preferably, the high-pressure homogenization is performed 3-4 times.

[0034] This invention has found that too many or too few high-pressure homogenization cycles can affect the drug loading.

[0035] Thirdly, the present invention provides the use of tofenamic acid liposomes according to the first aspect in the preparation of anti-inflammatory, antipyretic or analgesic drugs.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] Liposomes, as targeted drug carriers, can encapsulate various types of drugs, including both hydrophobic and hydrophilic drugs, through their hydrophobic layer and hydrophilic core, thereby improving drug solubility. Furthermore, liposomes have the potential to utilize the EPR (enhanced permeability and retention) effect. When liposomes enter the body, they accumulate near inflamed tissues with increased vascular permeability, more effectively delivering drugs to the lesion site and reducing drug toxicity. The targeting and sustained-release effects of liposomes can prolong the drug's half-life, improve the therapeutic index, and reduce the dosage required. Therefore, to enhance the water solubility of tofenamic acid, improve efficacy, and increase drug safety, tofenamic acid is encapsulated in liposomes. This improves the solubility of tofenamic acid and provides a high encapsulation efficiency and drug loading capacity. Attached Figure Description

[0038] Figure 1 This is a transmission electron microscope image of the tofenamic acid liposomes prepared in Example 1.

[0039] Figure 2 The particle size distribution of tofenamic acid liposomes prepared in Example 1 is shown in the diagram.

[0040] Figure 3 The potential diagram is for the tofenamic acid liposomes prepared in Example 1.

[0041] Figure 4Transmission electron microscopy image of tofenamic acid liposomes prepared in Example 11.

[0042] Figure 5 The particle size distribution of tofenamic acid liposomes prepared in Example 11 is shown in the diagram.

[0043] Figure 6 The potential diagram is for the tofenamic acid liposomes prepared in Example 11. Detailed Implementation

[0044] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0045] The sources of the active ingredients in the products described in the following examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0046] Hydrogenated phosphatidylcholine is a product of Sigma-Aldrich with product number 840058P.

[0047] The cholesterol is a product from Sigma, with product number C8667.

[0048] DSPE-PEG2000 is a product of Xi'an Ruixi Biotechnology Co., Ltd., with CAS number 474922-77-5.

[0049] Tofenamic acid is a product of McLean Company with product number T864371.

[0050] Example 1

[0051] This embodiment provides a tofenamic acid liposome, which comprises, by mass parts, 10.6 parts of hydrogenated phosphatidylcholine, 2.6 parts of cholesterol, 1 part of tofenamic acid, 0.5 parts of DSPE-PEG2000, and disodium succinate hexahydrate.

[0052] Its preparation method is as follows:

[0053] (1) Dissolve 10.6 mg of hydrogenated phosphatidylcholine, 2.6 mg of cholesterol, 0.5 mg of DSPE-PEG2000 and 1 mg of tofenamic acid in 4 mL of a mixed solution of methanol and chloroform (the volume ratio of methanol to chloroform is 1:1), heat at 37 °C and evaporate at 100 rpm. The remaining components form a thin film on the container wall.

[0054] (2) Add 3 mL of deionized water to the container in step (1), bathe in a water bath at 65°C for 20 min, and sonicate at 500 W power for 12 min.

[0055] (3) Add disodium succinate hexahydrate to the solution obtained in step (2) until the pH is 6.5;

[0056] (4) The liposome solution was obtained by homogenizing three times at 18,000 psi using a microfluidic high-pressure homogenizer (NanoGenizer30K).

[0057] Transmission electron microscopy image of the obtained tofenamic acid liposomes is shown below. Figure 1 As shown, by Figure 1 It can be seen that the tofenamic acid liposomes are evenly distributed and uniform in size, and are single-compartment liposomes, with tofenamic acid encapsulated in a lipid bilayer. Figure 2 and Figure 3 The particle size and potential diagrams of tofenamic acid liposomes prepared in Example 1 are shown below. Figure 2 and Figure 3 It is known that the average particle size of tofenamic acid liposomes is 105.3 nm, the polymer dispersibility index (PDI) is 0.2, and the zeta potential is -6.08 mV.

[0058] Example 2

[0059] This embodiment provides a tofenamic acid liposome, which comprises, by mass parts, 12 parts of hydrogenated phosphatidylcholine, 5 parts of cholesterol, 1.5 parts of tofenamic acid, 0.5 parts of DSPE-PEG2000, and disodium succinate hexahydrate.

[0060] Its preparation method is as follows:

[0061] (1) Dissolve 12mg of hydrogenated phosphatidylcholine, 5mg of cholesterol, 0.5mg of DSPE-PEG2000 and 1.5mg of tofenamic acid in 4mL of a mixed solution of methanol and chloroform (the volume ratio of methanol to chloroform is 1:1), heat at 36℃ and set the rotation speed to 90rpm for rotary evaporation. The remaining components form a thin film on the container wall.

[0062] (2) Add 3 mL of deionized water to the container in step (1), bathe in a water bath at 65°C for 15 min, and sonicate at 400 W power for 15 min.

[0063] (3) Add disodium succinate hexahydrate to the solution obtained in step (2) until the pH is 6;

[0064] (4) The liposome solution was obtained by homogenizing four times at 15000 psi using a microfluidic high-pressure homogenizer (NanoGenizer30K).

[0065] Example 3

[0066] This embodiment provides a tofenamic acid liposome, which comprises, by mass parts, 10 parts of hydrogenated phosphatidylcholine, 2 parts of cholesterol, 1 part of tofenamic acid, 0.5 parts of DSPE-PEG2000, and disodium succinate hexahydrate.

[0067] Its preparation method is as follows:

[0068] (1) Dissolve 10mg of hydrogenated phosphatidylcholine, 2mg of cholesterol, 0.5mg of DSPE-PEG2000 and 1mg of tofenamic acid in 4mL of a mixed solution of methanol and chloroform (the volume ratio of methanol to chloroform is 1:1), heat at 38°C, and evaporate at a speed of 110rpm. The remaining components form a thin film on the container wall.

[0069] (2) Add 3 mL of deionized water to the container in step (1), bathe in a water bath at 65°C for 20 min, and sonicate at 600 W for 10 min.

[0070] (3) Add disodium succinate hexahydrate to the solution obtained in step (2) until the pH is 7;

[0071] (4) The liposome solution was obtained by homogenizing four times at 20,000 psi using a microfluidic high-pressure homogenizer (NanoGenizer30K).

[0072] Example 4

[0073] This embodiment provides a tofenamic acid liposome, which differs from Example 1 only in that hydrogenated phosphatidylcholine is replaced with dimyristoyl phosphatidylcholine in equal amounts, while the contents of other components remain unchanged.

[0074] The preparation method is the same as in Example 1.

[0075] Example 5

[0076] This embodiment provides a tofenamic acid liposome, which differs from Example 1 only in that hydrogenated phosphatidylcholine is replaced with dioleoyl phosphatidylcholine in equal amounts, while the contents of other components remain unchanged.

[0077] The preparation method is the same as in Example 1.

[0078] Example 6

[0079] This embodiment provides a tofenamic acid liposome, which differs from Example 1 only in that hydrogenated phosphatidylcholine is replaced with distearate phosphatidylcholine in equal amounts, while the contents of other components remain unchanged.

[0080] The preparation method is the same as in Example 1.

[0081] Example 7

[0082] This embodiment provides a tofenamic acid liposome, which differs from Example 1 only in that step (4) of the preparation method is "homogenized once at 18000 psi using a microfluidic high-pressure homogenizer (NanoGenizer30K) to obtain a liposome solution". The composition and content of the tofenamic acid liposome remain unchanged, and other operations remain unchanged.

[0083] Example 8

[0084] This embodiment provides a tofenamic acid liposome, which differs from Example 1 only in that step (4) of the preparation method is "homogenized 5 times at 18000psi using a microfluidic high-pressure homogenizer (NanoGenizer30K) to obtain a liposome solution". The composition and content of the tofenamic acid liposome remain unchanged, and other operations remain unchanged.

[0085] Example 9

[0086] This embodiment provides a tofenamic acid liposome, which comprises, by mass parts, 10 parts of hydrogenated phosphatidylcholine, 1 part of cholesterol, 1 part of tofenamic acid, 0.5 parts of DSPE-PEG2000, and disodium succinate hexahydrate.

[0087] The preparation method is the same as in Example 1.

[0088] Example 10

[0089] This embodiment provides a tofenamic acid liposome, which comprises, by mass parts, 10 parts of hydrogenated phosphatidylcholine, 9 parts of cholesterol, 1 part of tofenamic acid, 0.5 parts of DSPE-PEG2000, and disodium succinate hexahydrate.

[0090] The preparation method is the same as in Example 1.

[0091] Example 11

[0092] This embodiment provides a tofenamic acid liposome, the raw materials for preparing the liposome include, by mass parts, 10.6 parts of hydrogenated phosphatidylcholine, 2.6 parts of cholesterol, 1 part of tofenamic acid, and disodium succinate hexahydrate.

[0093] The preparation method is the same as in Example 1.

[0094] Figure 4 Transmission electron microscopy image of tofenamic acid liposomes prepared in Example 11; by Figure 4 It can be seen that the tofenamic acid liposomes are uniformly distributed and are monocompartments, with tofenamic acid encapsulated in a lipid bilayer. Figure 5 and Figure 6 The particle size and potential diagrams of tofenamic acid liposomes prepared in Example 11 are shown below. Figure 5 and Figure 6It can be seen that the average particle size of tofenamic acid liposomes is 91.28 nm, the PDI is 0.5, and the zeta potential is -9.41 mV.

[0095] Compared with Example 1, the tofenamic acid liposomes obtained in Example 11 had a higher PDI and a worse stabilization effect, indicating that the addition of DSPE-PEG2000 is very important for maintaining the stability of liposomes.

[0096] Test Example 1

[0097] Encapsulation efficiency and drug loading detection

[0098] Test samples: Examples 1-11

[0099] Detection method: Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) was used for detection.

[0100] (1) Analysis method: Waters... T3 column (100×2.1mm, 1.8μm); mobile phase A was formic acid:water (0.001:1, v / v), and mobile phase B was formic acid:acetonitrile (0.001:1, v / v). Flow rate was 0.3 ml·min⁻¹; column temperature was 40℃.

[0101] Ultra-high performance liquid chromatography elution program:

[0102] Table 1

[0103] Washing time (min) Mobile phase A (%) Mobile phase B (%) 0.01 90 10 1.00 10 90 2.50 10 90 3.50 90 10 4.00 90 10

[0104] Mass spectrometry parameters: negative ion mode (ESI-) was used, quantitative ion pair was 260>216.2 m / z, qualitative ion pair was 260>180.1 m / z, and collision energy was 30 V.

[0105] (2) Sample pretreatment: Take 1 mL of tofenamic acid liposome solution, add 4 mL of acetonitrile, shake for 10 min, centrifuge at 8000 rpm for 15 min, take 1 mL of supernatant and detect it by ultra-high performance liquid chromatography-tandem mass spectrometry, and calculate the mass of tofenamic acid loaded into liposomes.

[0106] The encapsulation ratio formula is:

[0107] Encapsulation efficiency (%) = (mass of encapsulated tofenamic acid / initial mass of tofenamic acid) × 100%

[0108] The formula for drug loading is:

[0109] Drug loading (%) = (mass of tofenamic acid drug encapsulated / total initial input mass) × 100%

[0110] Table 2

[0111] sample Encapsulation rate Drug loading Example 1 78.2％ 5.3％ Example 2 75.4％ 4.6％ Example 3 73.9％ 4.9％ Example 4 42.5％ 2.9％ Example 5 57.2％ 3.9％ Example 6 40.1％ 2.7％ Example 7 60.1％ 4.1％ Example 8 32.7％ 2.2％ Example 9 62.3％ 3.4％ Example 10 58.7％ 2.9％ Example 11 89.2％ 5.6％

[0112] As shown in Table 2, the liposomes described in this invention have high encapsulation efficiency and drug loading capacity. The selection of phospholipids, the number of high-pressure homogenization cycles, and the mass ratio of phospholipids, cholesterol, and tofenamic acid are all very important for the encapsulation efficiency and drug loading capacity.

[0113] The applicant declares that this invention illustrates a tofenamic acid liposome, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A tofenamic acid liposome, characterized in that, The raw materials for preparing the liposomes include, by mass, 10-12 parts of phospholipids, 2-6 parts of cholesterol, and 1-1.5 parts of tofenamic acid, wherein the tofenamic acid is encapsulated in a phospholipid bilayer. The raw materials for preparing the liposomes also include a solubilizer, wherein the solubilizer is DSPE-PEG2000; The phospholipid is hydrogenated phosphatidylcholine; The tofenamic acid liposomes were prepared by a method comprising the following steps: (1) Mix phospholipids, cholesterol, tofenamic acid with an organic solvent and rotary evaporate until a thin film forms on the container wall; (2) Add deionized water to the container in step (1) and sonicate to obtain a dispersion; (3) The dispersion obtained in step (2) is homogenized under high pressure to obtain the final product; The high-pressure homogenization is performed 3-4 times.

2. The tofenamic acid liposome according to claim 1, characterized in that, The pH of the liposomes is 6-7.

3. The method for preparing tofenamic acid liposomes according to claim 1 or 2, characterized in that, The preparation method includes: (1) Mix phospholipids, cholesterol, tofenamic acid with an organic solvent and rotary evaporate until a thin film forms on the container wall; (2) Add deionized water to the container in step (1) and sonicate to obtain a dispersion; (3) The dispersion obtained in step (2) is homogenized under high pressure to obtain the final product; The high-pressure homogenization is performed 3-4 times.

4. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, Step (2) also includes mixing the dispersion with a pH adjuster.

5. The method for preparing tofenamic acid liposomes according to claim 4, characterized in that, The pH adjuster includes disodium succinate hexahydrate.

6. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, The organic solvents include methanol and / or chloroform.

7. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, The rotary evaporator operates at a temperature of 36-38°C and a rotation speed of 90-110 rpm.

8. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, The process of adding deionized water also includes a 60-65℃ water bath for 15-25 minutes.

9. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, The ultrasonic power is 400-600 W, and the duration is 10-15 min.

10. The method for preparing tofenamic acid liposomes according to claim 3, characterized in that, The high-pressure homogenization is performed at a pressure of 15,000-20,000 psi for 3-5 seconds.

11. The use of tofenamic acid liposomes according to claim 1 or 2 in the preparation of anti-inflammatory, antipyretic or analgesic drugs.

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