An entecavir liposome, preparation and preparation method thereof

Abstract

The present invention relates to an entecavir liposome, a preparation thereof and a preparation method thereof, belonging to the technical field of antiviral drug preparations. The entecavir liposome of the present invention comprises entecavir, phosphatidylcholine, cholesterol, phosphatidylserine, and calcium lignosulfonate. The entecavir liposome of the present invention is obtained through film preparation, hydration, and liposome preparation. The present invention optimizes the formulation of the entecavir liposome, improves the stability of the entecavir liposome, enables the entecavir liposome to maintain the encapsulated state of the drug during storage and transportation, reduces the leakage of the drug, and thus improves the stability of the preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral pharmaceutical preparations, and particularly relates to an entecavir liposome, a preparation thereof and a preparation method therefor. Background Art

[0002] Entecavir is an antiviral drug widely used in the treatment of chronic hepatitis B (HBV). Since its approval by the US FDA in 2005, entecavir has become a commonly used drug for the treatment of chronic hepatitis B. Its mechanism of action is that entecavir is converted into its active metabolite entecavir triphosphate (ETV-TP) in vivo. ETV-TP inhibits the activity of hepatitis B virus (HBV) reverse transcriptase through competition, preventing the virus's RNA from being transcribed into DNA, and thus inhibiting virus replication. It mainly acts through the following mechanisms: (1) Termination of chain elongation: EVA-TP binds to the HBV DNA strand, resulting in the termination of the viral DNA strand; (2) Reverse transcriptase inhibition: EVA-TP can directly inhibit the reverse transcriptase of HBV, interfering with the process of transcribing RNA into DNA; (3) Reduction of virus load: Long-term use of entecavir can significantly reduce the level of HBV DNA in the blood, thereby alleviating the inflammatory and fibrotic processes of the liver.

[0003] The clinical uses of entecavir mainly focus on the treatment of hepatitis B, specifically including: (1) Treatment of chronic hepatitis B: Entecavir can significantly inhibit the replication of hepatitis B virus, reduce liver damage, and improve liver function; (2) Management of hepatitis B cirrhosis: For hepatitis B patients with cirrhosis, entecavir can reduce virus replication, improve liver function, and delay the progression of cirrhosis; (3) Treatment of acute exacerbations caused by hepatitis B virus: In some hepatitis B patients with acute exacerbations, entecavir, as one of the options for antiviral treatment, helps to control the condition.

[0004] Entecavir liposome drug is a novel drug delivery system that improves the traditional entecavir drug based on liposome technology. Liposome is a micro-spherical structure formed by phospholipid molecules, which can encapsulate drugs therein, enhance the stability of drugs, improve their bioavailability and control drug release.

[0005] The Chinese patent with the publication number CN105534905A discloses an entecavir multi-lamellar liposome, which includes entecavir, glyceryl trioleate, neutral phospholipids, cholesterol, acid regulators, osmotic pressure regulators, and auxiliary emulsifiers. The Chinese patent with the publication number CN105560216A discloses an entecavir solid lipid nanoparticle, which includes entecavir, poloxamer 188, tween-80, glyceryl monostearate, etc. The Chinese patent with the publication number CN105412015A discloses a glycyrrhetinic acid-modified entecavir liposome preparation, which is prepared by modifying phospholipids with glycyrrhetinic acid and polyethylene glycol.

[0006] The entecavir liposomes in the prior art have the problem of poor stability. When placed at 25 °C for one month, the content decreases by nearly 3%, and the particle size increases by 12 nm. This may cause the rupture of the liposome membrane or the degradation of the drug, resulting in the inability to continuously release the drug, thereby increasing side effects or toxicity. Summary of the Invention

[0007] To overcome the deficiencies of the prior art, the present invention provides an entecavir liposome, a preparation thereof, and a preparation method. This method optimizes the formula of the entecavir liposome, and the prepared entecavir liposome and preparation have high stability.

[0008] The entecavir liposome of the present invention is composed of 1 to 3 parts by weight of entecavir, 1 to 2 parts by weight of phosphatidylcholine, 0.2 to 0.8 parts by weight of cholesterol, 0.5 to 1.5 parts by weight of phosphatidylserine, and 0.05 to 0.2 parts by weight of calcium lignosulfonate.

[0009] The preparation method of the above entecavir liposome includes the following steps:

[0010] (1) Film preparation: Dissolve entecavir, phosphatidylcholine, cholesterol, and phosphatidylserine in a mixed solvent, and heat and rotate to evaporate in a water bath at 35 °C to 45 °C to form a film;

[0011] (2) Hydration: Dissolve calcium lignosulfate (CAS: 8061-52-7) in a buffer solution with a pH of 4.5 to 6.0, add it to the film obtained in step (1) for hydration, and heat and rotate to evaporate in a water bath at 40 °C to 50 °C for 10 to 20 min to obtain solution A;

[0012] (3) Liposome preparation: Subject solution A in step (2) to high-pressure homogenization, filtration, and freeze-drying to obtain entecavir liposomes.

[0013] Preferably, the mixed solvent in step (1) is 2,6-nonadienol and methanol with a volume ratio of 1:2 to 5 or 2,6-nonadienol and ethanol with a volume ratio of 1:2 to 5.

[0014] Preferably, the buffer solution in step (2) is any one of acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution, and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution.

[0015] Preferably, the freeze-drying procedure in step (3) is as follows: a. Pre-freezing: temperature -35°C, time 2 h; b. Sublimation drying: temperature -20°C, pressure <100 Pa, time 12 h; c. Desorption drying: temperature 30°C, pressure <100 Pa, time 6 h; d. Sealing.

[0016] The present invention also provides a preparation composed of the above-mentioned entecavir liposome and pharmaceutically acceptable excipients, including but not limited to tablets, granules, capsules, and oral liquids. The pharmaceutically acceptable excipients are fillers, lubricants, wetting agents, disintegrants, binders, preservatives, flavoring agents, surfactants, solvents, and coating agents.

[0017] Compared with the prior art, the technical effects of the present invention are as follows:

[0018] The present invention has screened out a suitable solvent, and uses a mixed solvent of 2,6-nonadienol and ethanol or methanol to dissolve lipid materials to prepare liposomes, which can regulate the fluidity of the liposome membrane and can significantly improve the entrapment efficiency and stability of entecavir liposomes.

[0019] The present invention has optimized the formulation of entecavir liposomes. Especially the addition of calcium lignosulfate has improved the stability of liposomes to a certain extent, enabling entecavir liposomes to maintain the drug-entrapped state during storage and transportation, reducing drug leakage, and thus improving the stability of the preparation. Description of the Drawings

[0020] Figure 1 It is the entrapment efficiency of entecavir liposomes on the 1st day of the stability experiment at 25°C;

[0021] Figure 2 It is the entrapment efficiency of entecavir liposomes on the 30th day of the stability experiment at 25°C;

[0022] Figure 3 It is the drug loading of entecavir liposomes obtained in Examples 1-3 and Comparative Examples 1-3;

[0023] Figure 4 It is the entrapment efficiency of entecavir liposomes obtained in Examples 1-3 and Comparative Examples 1-3. Detailed Embodiments

[0024] In order to make the objectives and technical solutions of the present invention more clear and understandable, the following further describes the present invention in conjunction with 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.

[0025] Solvent screening

[0026] In the preparation of liposomal drugs, the type and proportion of solvents have an important impact on the properties and quality of liposomes.

[0027] A: 2,6-Nonadienol and methanol with a volume ratio of 1:4

[0028] B: 2,6-Nonadienol and ethanol with a volume ratio of 1:2

[0029] C: 2,6-Nonadienol and ethanol with a volume ratio of 1:5

[0030] D: Methanol

[0031] E: Ethanol

[0032] F: Chloroform

[0033] G: 2,6-Nonadienol and methanol with a volume ratio of 5:1

[0034] H: 2,6-Nonadienol

[0035] I: 2,6-Nonadienol and chloroform with a volume ratio of 1:4

[0036] Based on the preparation of entecavir liposomes in Example 1, the effect of solvent selection on entecavir liposomes was explored.

[0037] Figure 1 Shows the encapsulation efficiency of entecavir liposomes on the first day of the stability experiment at 25°C, Figure 2 Shows the encapsulation efficiency of entecavir liposomes on the 30th day of the stability experiment at 25°C. The experimental data show that the solvent selection has an important impact on the encapsulation efficiency and stability of entecavir liposomes. Generally speaking, chloroform, methanol, and ethanol are commonly used solvents for liposome preparation, which have good solubility and can dissolve various lipid components such as phospholipids and cholesterol, enabling the lipid components to be fully mixed and facilitating the formation of a uniform lipid membrane. It was found in the experiment that the encapsulation efficiency of entecavir liposomes prepared with the above-mentioned common solvents or combinations in the present invention is not high and the stability is average, while the mixed solvent formed by mixing 2,6-nonadienol and ethanol or methanol in a certain proportion can improve the encapsulation efficiency and stability of entecavir liposomes. 2,6-Nonadienol may interact with the membrane components of liposomes to regulate the fluidity and stability of the liposome membrane.

[0038] Table 1 Particle Sizes of Entecavir Liposomes Prepared with Different Solvents

[0039]

[0040] From the perspective of particle size, the influence of solvents on entecavir liposomes was explored. It was found that solvents can affect the formation process and kinetics of entecavir liposomes, thereby changing the particle size and particle size distribution. The mixed solvent of 2,6-nonadienol and methanol or ethanol can make the particle size distribution more concentrated and uniform. At the same time, when the stability experiment was carried out at 25 °C for 30 hours, the particle size of entecavir liposomes changed little and had high stability.

[0041] Example 1

[0042] A preparation method of entecavir liposomes

[0043] (1) Film preparation: Dissolve 2.0 g of entecavir, 1.5 g of phosphatidylcholine, 0.4 g of cholesterol, and 1.0 g of phosphatidylserine in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:3, and heat and rotate for evaporation in a water bath at 40 °C to form a film;

[0044] (2) Hydration: Dissolve 0.12 g of calcium lignosulfate in a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 5.2, add it to the film obtained in step (1) for hydration, and heat and rotate for evaporation in a water bath at 45 °C for 15 min to obtain solution A;

[0045] (3) Liposome preparation: Subject solution A in step (2) to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35 °C, time 2 h; b. Sublimation drying: temperature -20 °C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30 °C, pressure < 100 Pa, time 6 h; d. Sealing) to obtain entecavir liposomes with an average particle size of 310.24 nm ± 5.33 nm.

[0046] A formulation of entecavir tablets:

[0047] The above-mentioned entecavir liposomes (containing 0.5 mg of entecavir), 150 mg of starch, 50 mg of microcrystalline cellulose, 5 mg of magnesium stearate, 12 mg of sodium carboxymethyl starch, and Opadry coating premix.

[0048] Preparation method:

[0049] After sieving the above-mentioned raw and auxiliary materials, mix the entecavir liposomes, starch, and microcrystalline cellulose, add sodium carboxymethyl starch and magnesium stearate, mix evenly, press into tablets, and coat the tablets (weight gain 1.5% - 3.5%) to obtain the tablets.

[0050] A formulation of entecavir oral solution:

[0051] The above entecavir liposome (containing 0.5 mg of entecavir), water, 1 mg of sucrose, 1 mg of glycerol, 0.2 mg of poloxamer 188, and 0.02 mg of ethylparaben.

[0052] Preparation method:

[0053] Add glycerol, an appropriate amount of water, and poloxamer to the above entecavir liposome for dissolution, add ethylparaben and sucrose, and after complete dissolution, add water to 20 mL to obtain the product.

[0054] Example 2

[0055] A preparation method of entecavir liposome

[0056] (1) Film preparation: Dissolve 1.0 g of entecavir, 1.0 g of phosphatidylcholine, 0.2 g of cholesterol, and 0.5 g of phosphatidylserine in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:2, and perform rotary evaporation by heating in a water bath at 35 °C to form a film;

[0057] (2) Hydration: Dissolve 0.05 g of calcium lignosulfate in a citric acid-sodium citrate buffer solution with a pH of 4.5, add it to the film obtained in step (1) for hydration, and perform rotary evaporation by heating in a water bath at 40 °C for 20 min to obtain solution A;

[0058] (3) Liposome preparation: Subject solution A in step (2) to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35 °C, time 2 h; b. Sublimation drying: temperature -20 °C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30 °C, pressure < 100 Pa, time 6 h; d. Sealing) to obtain entecavir liposome with an average particle size of 362.50 nm ± 7.02 nm.

[0059] A formulation of entecavir granules:

[0060] The above entecavir liposome (containing 0.5 mg of entecavir), 100 mg of starch, 40 mg of sucrose, 50 mg of dextrin, 2 mg of colloidal silica, 8 mg of low-substituted hydroxypropyl methylcellulose, and an appropriate amount of water.

[0061] Preparation method:

[0062] Sieve the above raw and auxiliary materials, mix the entecavir liposome, starch, sucrose, dextrin, colloidal silica, and low-substituted hydroxypropyl methylcellulose, add an appropriate amount of water, granulate, size the granules, and dry to obtain the granules.

[0063] A formulation of entecavir capsules:

[0064] The above entecavir liposome (containing 0.5 mg of entecavir), 200 mg of starch, 50 mg of lactose, 2 mg of talc powder, 5 mg of crospovidone, and capsule shell.

[0065] Preparation method:

[0066] The above raw and auxiliary materials are sieved, and the entecavir liposome, starch, lactose, talc powder, and crospovidone are mixed, filled into the capsule shell, and sealed to obtain the product.

[0067] Example 3 Entacavir Liposome and Its Preparation

[0068] (1) Film preparation: Dissolve 3.0 g of entecavir, 2.0 g of phosphatidylcholine, 0.8 g of cholesterol, and 1.5 g of phosphatidylserine in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:5, and heat and rotate-evaporate in a 45°C water bath to form a film;

[0069] (2) Hydration: Dissolve 0.2 g of calcium lignosulfate in an acetic acid-sodium acetate buffer solution with pH = 6.0, add it to the film obtained in step (1) for hydration, and heat and rotate-evaporate in a 50°C water bath for 10 min to obtain solution A;

[0070] (3) Liposome preparation: Subject solution A in step (2) to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35°C, time 2 h; b. Sublimation drying: temperature -20°C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30°C, pressure < 100 Pa, time 6 h; d. Sealing) to obtain entecavir liposome with an average particle size of 342.85 nm ± 6.78 nm.

[0071] Comparative Example 1

[0072] A preparation method of entecavir liposome

[0073] (1) Film preparation: Dissolve 2.0 g of entecavir, 1.5 g of phosphatidylcholine, 0.4 g of cholesterol, and 1.0 g of phosphatidylserine in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:3, and heat and rotate-evaporate in a 40°C water bath to form a film;

[0074] (2) Hydration: Prepare a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with pH = 5.2, add it to the film obtained in step (1) for hydration, and heat and rotate-evaporate in a 45°C water bath for 15 min to obtain solution A;

[0075] (3) Liposome preparation: The solution A in step (2) was subjected to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35°C, time 2 h; b. Sublimation drying: temperature -20°C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30°C, pressure < 100 Pa, time 6 h; d. Sealing), to obtain entecavir liposomes with an average particle size of 392.17 nm ± 16.05 nm.

[0076] Comparative Example 2

[0077] A method for preparing entecavir liposomes (1) Film preparation: 2.0 g of entecavir, 1.5 g of phosphatidylcholine, 0.4 g of cholesterol, and 1.0 g of phosphatidylserine were dissolved in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:3, and rotary evaporation was carried out by heating in a water bath at 40°C to form a film;

[0078] (2) Hydration: 0.12 g of poloxamer 188 was dissolved in a sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution with pH = 5.2, added to the film obtained in step (1) for hydration, and rotary evaporation was carried out by heating in a water bath at 45°C for 15 min to obtain solution A;

[0079] (3) Liposome preparation: The solution A in step (2) was subjected to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35°C, time 2 h; b. Sublimation drying: temperature -20°C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30°C, pressure < 100 Pa, time 6 h; d. Sealing), to obtain entecavir liposomes with an average particle size of 374.15 nm ± 12.30 nm.

[0080] Comparative Example 3

[0081] A method for preparing entecavir liposomes (1) Film preparation: 2.0 g of entecavir, 1.5 g of phosphatidylcholine, 0.4 g of cholesterol, and 1.0 g of phosphatidylserine were dissolved in 50 mL of a mixed solvent of 2,6-nonadienol and methanol with a volume ratio of 1:3, and rotary evaporation was carried out by heating in a water bath at 40°C to form a film;

[0082] (2) Hydration: 0.50 g of calcium lignosulfate was dissolved in a sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution with pH = 5.2, added to the film obtained in step (1) for hydration, and rotary evaporation was carried out by heating in a water bath at 45°C for 15 min to obtain solution A;

[0083] (3)Liposome preparation: The solution A in step (2) was subjected to high-pressure homogenization, filtration, and freeze-drying (a. Pre-freezing: temperature -35°C, time 2 h; b. Sublimation drying: temperature -20°C, pressure < 100 Pa, time 12 h; c. Desorption drying: temperature 30°C, pressure < 100 Pa, time 6 h; d. Sealing), to obtain entecavir liposomes with an average particle size of 442.61 nm ± 18.48 nm.

[0084] Test Example 1

[0085] Quality evaluation of entecavir liposomes:

[0086] Drug loading of entecavir liposomes

[0087] The drug loading is the mass percentage (g / g) of the drug contained in the liposomes, and the drug loading = the amount of drug contained in the liposomes / the total amount of liposomes × 100%.

[0088] Figure 3 is the drug loading of entecavir liposomes. The drug loading of liposomes determines the effective concentration of entecavir. Experiments show that Examples 1 - 3 have a higher drug loading, which means that more drugs can be carried in liposome preparations of the same dose, so that a higher effective drug concentration can be achieved at the site of action after administration, enhancing the therapeutic effect.

[0089] Entrapment efficiency of entecavir liposomes

[0090] After measuring the total amount of drug in the liposomes, it was separated by a chromatographic column, and the amount of drug not encapsulated in the medium was measured. The entrapment efficiency = 1 - the amount of drug not encapsulated in the liquid medium / the total amount of encapsulated and unencapsulated drugs in the liposomes × 100%. Generally, the entrapment efficiency of liposomes should not be less than 80%.

[0091] Figure 4 is the entrapment efficiency of entecavir liposomes. The entrapment efficiency is one of the important indicators to measure the quality of liposome preparations. The entecavir liposomes in Examples 1 - 3 of the present invention have a higher entrapment efficiency, which means that more drugs are effectively encapsulated in the liposomes, further indicating that the quality of the prepared preparations is higher.

[0092] Leakage rate of entecavir liposomes

[0093] The change in the entrapment efficiency of liposomes during storage is the main indicator of liposome stability. The liposomes were stored at 25°C, sampled on the 60th day, and the amount of drug encapsulated or free drug in the liposomes was measured, so as to obtain the amount of drug leaked into the medium after storage. Compared with the amount of drug encapsulated before storage, the leakage rate = the amount of drug leaked into the medium after a certain storage time / the amount of drug encapsulated before storage × 100%.

[0094] Table 2 Leakage rate of entecavir liposomes (%)

[0095]

[0096] Table 2 shows the leakage of entecavir liposomes stored at 25°C for 60 days. The low leakage rates of the entecavir liposomes in Examples 1 to 3 mean that the liposomes can maintain the drug encapsulation state during storage and transportation, reduce drug leakage, and thus improve the stability of the preparation.

Claims

1. An entecavir liposome, characterized in that: Composed of 1-3 parts by weight of entecavir, 1-2 parts by weight of phosphatidylcholine, 0.2-0.8 parts by weight of cholesterol, 0.5-1.5 parts by weight of phosphatidylserine, and 0.05-0.2 parts by weight of calcium lignin sulfonate; The method for preparing the entecavir liposomes comprises the following steps: (1) Film preparation: Entecavir, phosphatidylcholine, cholesterol, and phosphatidylserine are dissolved in a mixed solvent, heated in a water bath at 35°C to 45°C and subjected to rotary evaporation to form a film; the mixed solvent is 2,6-nonadienol and methanol or ethanol in a volume ratio of 1:2 to 5; (2) Hydration: Dissolve calcium lignin sulfonate (CAS: 8061-52-7) in a buffer solution with a pH of 4.5 to 6.0, add the solution to the film obtained in step (1) for hydration, and heat the solution in a water bath at 40°C to 50°C for 10 to 20 min by rotary evaporation to obtain a solution A. (3) Preparation of liposomes: Solution A in step (2) is homogenized under high pressure, filtered, and freeze-dried to obtain entecavir liposomes.

2. The entecavir liposome according to claim 1, characterized in that: The buffer solution in step (2) is any one of acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution, and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution.

3. The entecavir liposome according to claim 1, characterized in that: The pH of the buffer solution in step (2) is 5.

2.

4. The entecavir liposome according to claim 1, characterized in that: The freeze-drying procedure in step (3) is as follows: a. pre-freezing: temperature -35°C, time 2h, b. sublimation drying: temperature -20°C, pressure <100Pa, time 12h, c. desorption drying: temperature 30°C, pressure <100Pa, time 6h, d. sealing.

5. The entecavir liposome according to claim 1, characterized in that: The particle size of the entecavir liposome is less than 500 nm.

6. A preparation, characterized in that The preparation consists of the entecavir liposomes according to claim 1 and pharmaceutically acceptable excipients.

7. The preparation according to claim 6, characterized in that The pharmaceutically acceptable excipients are selected from fillers, lubricants, wetting agents, disintegrants, binders, preservatives, flavoring agents, surfactants, solvents, and coating agents.

8. The preparation according to claim 6, characterized in that The preparation is in the form of tablets, granules, capsules and oral liquid.

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