CN105853361B - A kind of tetrazine dicarboxamide liposome preparation and preparation method thereof - Google Patents

Abstract

The invention discloses a tetrazine dicarboxamide liposome preparation and a preparation method thereof. The components of the preparation and their weight parts are: tetrazine dicarboxamide: 1.0-2.0 parts; phospholipid: 12.5-62.5 parts; Cholesterol: 2.5-6.25 parts; Vitamin E: 0-2.0 parts; Organic solvent: 2.5-7.5 parts; Phosphate buffer solution: 3000-11,000 parts. The phospholipid material is selected from one of lecithin, soybean lecithin, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and sphingomyelin; the organic solvent is selected from one of ethanol, ether, chloroform, and dichloromethane: The pH value of the phosphate buffer solution is selected between 6.4 and 7.6. In the present invention, the liposome solution is prepared by a film dispersion method or an injection method, and the method of vacuum drying or constant temperature heating and stirring is used to remove the organic solvent in the drug solution in the preparation process, thereby avoiding the residue of the organic solvent.

Description

A kind of tetrazine dicarboxamide liposome preparation and preparation method thereof

technical field

The invention belongs to the technical field of pharmaceutical preparations, and relates to a preparation method of a new dosage form of tetrazine dicarboxamide, in particular to a tetrazine dicarboxamide liposome preparation and a preparation method thereof.

Background technique

Tetrazine, also known as tetraazabenzene, is a class of antitumor compounds with good biological activity. Tetrazine dicarboxamide (ZGDHu21) was synthesized by Hu Weixiao and others. It is a new type of tetrazine compound with independent intellectual property rights. It can It inhibits the proliferation of leukemia cells, lung cancer cells, liver cancer cells, gastric cancer cells, etc., and can play a certain role in inducing differentiation and apoptosis. In vivo activity studies in nude mice show that tetrazine dicarboxamide has good therapeutic activity on lung cancer. Statistics show that lung cancer is the malignant tumor with the highest morbidity and mortality among men. The structural formula of tetrazine dicarboxamide is shown in Figure 1.

Tumors are usually divided into benign tumors and malignant tumors. Among them, malignant tumors are often referred to as cancers, and there are more than 100 related diseases. Cancer is a disease of the body in which normal cells are mutated and continue to grow and divide without the control of the body. In today's society, the mortality rate of cancer ranks second among various diseases, second only to cardiovascular and cerebrovascular diseases. Due to the continuous increase of environmental pollution in modern society and the irregular living habits of people, the incidence of cancer is very high, and it will become another major killer of human beings.

Liposomes are spherical closed vesicles formed by dispersing phospholipids in water and encapsulating part of the inner water phase. In the 1970s, Gregoriadis and other scholars first proposed that liposomes could be used in the delivery of anti-tumor drugs, creating a new field of research on anti-cancer drug drug delivery systems. Liposomes can encapsulate water-soluble or fat-soluble drugs and change the kinetics and distribution properties of drugs, so their research and development has become a very active field.

Liposomes have many advantages as drug carriers. Due to the characteristics of its composition and structure, liposomes can encapsulate hydrophilic drugs in the inner aqueous phase and lipophilic drugs in phospholipid bilayers; liposomes can increase drug solubility, and Biofilms have good compatibility and low toxicity; after intravenous injection, they can delay drug release, improve bioavailability, and have a targeting effect; liposomes have achieved great success as carriers of antitumor drugs.

Influenced by the dissolution characteristics of tetrazine dicarboxamide (it has strong fat solubility and poor water solubility), the development of common preparation products of tetrazine dicarboxamide is limited, and the compound has been available for a short time, and the research on its preparation has not been reported yet. The preparation of tetrazine dicarboxamide into a new liposome dosage form can improve its water solubility, increase its bioavailability and make it have certain targeting properties.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defect that the drugs in the prior art cannot be absorbed in the body and are retained. The present invention provides a tetrazine dicarboxamide liposome preparation and a preparation method thereof. Using the principle of similar compatibility, the drug is encapsulated in fat-soluble vesicles, so that the water solubility of the drug is increased, and the biological activity can be produced in the body. It has a certain targeting effect and can be used clinically as an intravenous or intramuscular injection.

For realizing the purpose of the present invention, the concrete technical scheme is:

A tetrazine dicarboxamide liposome preparation, the components and the weight parts thereof are: tetrazine dicarboxamide: 1.0-2.0 parts; phospholipids: 12.5-62.5 parts; cholesterol: 2.5-6.25 parts; vitamin E: 0 to 2.0 parts; organic solvent: 2.5 to 7.5 parts; phosphate buffer solution: 3000 to 11000 parts.

The phospholipid material is selected from one of lecithin, soybean phospholipid, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and sphingomyelin. Preferably, the selected phospholipid material is lecithin (PC content 98%). Lecithin is a natural surfactant, a component of cell membranes, and has good affinity with the body, and is a commonly used phospholipid material in the preparation of liposomes. In the preparation process of tetrazine dicarboxamide liposome, the solution prepared by selecting lecithin has small particle size, small polydispersity coefficient and high encapsulation efficiency.

The described organic solvent is selected from one of ethanol, ether, chloroform, and dichloromethane, and preferably, the selected organic solvent is ethanol. Ethanol can better dissolve auxiliary materials such as drugs and phospholipids, and the prepared tetrazine dicarboxamide liposome solution is relatively clear, with small particle size, and ethanol is less toxic, less harmful to the environment and operators, and easy to remove.

The phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection. Using the phosphate buffer solution of pH 6.8, the prepared tetrazine dicarboxamide liposome solution is relatively stable, is not easy to precipitate, the solution particle size and polydispersity coefficient are small, and the solution is relatively clear.

Cholesterol is a natural amphiphilic substance. The main purpose of adding cholesterol in the prescription is to improve the stability of liposomes and reduce drug leakage. During the preparation process, too little or too much cholesterol is not conducive to the stability of liposomes. The solution particle size and polydispersity coefficient both increased. Vitamin E is an antioxidant that is added to the prescription to protect drugs and other excipients from oxidative denaturation.

A preparation method of tetrazine dicarboxamide liposome preparation, specifically comprises the following steps:

(1) Add tetrazine dicarboxamide, phospholipid, cholesterol, and vitamin E to an organic solvent, stir or ultrasonically treat it to completely dissolve it, and prepare solution A.

(2) Preparation by thin film dispersion method: Put solution A in a flask, remove the organic solvent by rotary evaporation, form a dry film on the bottle wall, add phosphate buffer solution to the flask, hydrate with ultrasonic probe, and filter through 0.45um microporous filter. Membrane filtration to obtain a liposome solution.

(3) Preparation by injection method: The solution A can also be injected into the continuously stirred phosphate buffer solution through a syringe to obtain a mixed solution. , filtered through a 0.45um microporous membrane to obtain a liposome solution.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention prepares tetrazine dicarboxamide into liposomes, which better solves the problem of poor water solubility of drugs, which is unfavorable for administration and absorption, and the prepared liposome solution has small particle size and low polydispersity coefficient. High encapsulation efficiency and good stability. Due to the particularity of its structure, the drug has a targeted effect, prolongs the action time in the body, improves the bioavailability, and is beneficial to the treatment of diseases.

(2) The selected auxiliary materials such as phospholipids and cholesterol in the present invention are convenient and easy to obtain, have good affinity with the body, and have low toxicity. The selection of excipients is also beneficial to the preparation of the liposome solution, which makes the preparation process simpler and easier to operate.

(3) The tetrazine dicarboxamide liposomes are prepared by the conventional film dispersion method and injection method. The appearance of the product is good, the solution is relatively clear, slightly milky white, and the preparation process is fast and simple.

(4) In the present invention, tetrazine dicarboxamide is made into a liposome solution, and the average particle size of the prepared solution is below 400 nm, the polydispersity coefficient is below 0.4, the drug encapsulation rate is above 60%, and the water solubility, Stability has been improved for better absorption by the body.

Description of drawings

Fig. 1 is the chemical structure of tetrazine dicarboxamide;

Fig. 2 is the particle size distribution figure of the tetrazine dicarboxamide liposome solution that the embodiment of the present invention 1 obtains;

Fig. 3 is the particle size distribution figure of the tetrazine dicarboxamide liposome solution that the embodiment of the present invention 2 obtains;

Fig. 4 is the particle size distribution diagram of the tetrazine dicarboxamide liposome solution obtained in Example 3 of the present invention;

Fig. 5 is the particle size distribution diagram of the tetrazine dicarboxamide liposome solution obtained in Example 4 of the present invention;

Fig. 6 is the particle size distribution diagram of the tetrazine dicarboxamide liposome solution obtained in Example 5 of the present invention;

Fig. 7 is the particle size distribution diagram of the tetrazine dicarboxamide liposome solution that the embodiment of the present invention 6 obtains;

Fig. 8 is the scanning electron microscope picture of the tetrazine dicarboxamide liposome solution obtained in Example 3 of the present invention;

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

Example 1:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 4 mg;

Cholesterol: 20mg;

Lecithin (PC content 98%): 100mg;

Ethanol: 10mL;

Phosphate buffer solution (pH 6.8): 20 mL.

Dissolve the recipe quantities of tetrazine dicarboxamide, lecithin and cholesterol in ethanol, stir and dissolve to obtain solution A; place solution A in a flask, use a rotary evaporator to remove the organic solvent ethanol, and the rotary evaporator constant temperature water bath is 45 ℃, the rotation speed is 100r/min, the vacuum degree is greater than 0.09MPa, and the bottle wall forms a dry film, and the evaporation is continued until the solvent is completely evaporated. The prescribed amount of phosphate buffer solution was added to the flask, the probe was ultrasonicated for 10 minutes, and the solution was hydrated. The solution was filtered with a 0.45um microporous membrane to obtain a slightly milky white drug liposome solution.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 107.0 nm, a polydispersity coefficient of 0.149, and a drug encapsulation rate of 78.2%.

Example 2:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 4 mg;

Cholesterol: 15mg;

Lecithin (PC content 98%): 150mg;

Vitamin E: 2mg;

Diethyl ether: 15mL;

Phosphate buffer solution (pH 6.4): 25 mL.

Dissolve the recipe quantities of tetrazine dicarboxamide, lecithin, cholesterol, and vitamin E in ether, stir and dissolve to obtain solution A; inject solution A into the phosphate buffer solution that is continuously stirred through a syringe to obtain a milky white mixture The mixed solution was placed in a 55°C constant temperature water bath, stirred continuously, volatilized to remove the organic solvent, and then filtered with a 0.45um microporous membrane to obtain a slightly clear milky white drug liposome solution.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 105.4 nm, a polydispersity coefficient of 0.152, and a drug encapsulation rate of 74.9%.

Example 3:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 8 mg;

Cholesterol: 10mg;

Soybean lecithin (PC content 70%): 80mg;

Vitamin E: 6mg;

Chloroform: 25mL;

Phosphate buffer solution (pH 7.0): 12 mL.

Dissolve the recipe amounts of tetrazine dicarboxamide, lecithin, cholesterol, and vitamin E in chloroform, and ultrasonically dissolve to obtain solution A; place solution A in a flask, use a rotary evaporator to remove the organic solvent ethanol, and keep the rotary evaporator at a constant temperature The water bath is 40°C, the speed is 100r/min, and the vacuum degree is greater than 0.09MPa. After the bottle wall forms a dry film, continue to evaporate until the solvent evaporates completely. The prescribed amount of phosphate buffer solution was added to the flask, the probe was ultrasonicated for 10 minutes, and the solution was hydrated. The solution was filtered with a 0.45um microporous membrane to obtain a slightly milky white drug liposome solution.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 337.9 nm, a polydispersity coefficient of 0.313, and a drug encapsulation rate of 68.4%.

Example 4:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 6mg;

Cholesterol: 25mg;

Sphingomyelin: 200mg;

Vitamin E: 8mg;

Dichloromethane: 20mL;

Phosphate buffer solution (pH 7.6): 40 mL.

Dissolve tetrazine dicarboxamide, sphingomyelin, cholesterol, and vitamin E in the prescribed amounts in dichloromethane, and ultrasonically dissolve to obtain solution A; inject solution A into a continuously stirred phosphate buffer solution through a syringe to obtain milky white The mixed solution was placed in a 50°C constant temperature water bath, stirred continuously, volatilized to remove the organic solvent, and then filtered with a 0.45um microporous membrane to obtain a slightly milky white drug liposome solution.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 168.8 nm, a polydispersity coefficient of 0.092, and a drug encapsulation rate of 75.1%.

Example 5:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 5mg;

Cholesterol: 12mg;

Phosphatidylserine: 50mg;

Diethyl ether: 30mL;

Phosphate buffer solution (pH 7.2): 44 mL.

The specific preparation method of the tetrazine dicarboxamide liposome in this example is the same as the method in Example 1, the only difference is that the raw materials and dosages used in this example are used, and the constant temperature water bath of the rotary evaporator is stable at 30 ° C , which will not be repeated here. A slightly milky white drug liposome solution was prepared.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 212.8 nm, a polydispersity coefficient of 0.314, and a drug encapsulation rate of 69.7%.

Example 6:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 6.5 mg;

Cholesterol: 22mg;

Phosphatidylglycerol: 250mg;

Vitamin E: 5mg

Ethanol: 28mL;

Phosphate buffer solution (pH 6.8): 30 mL.

The specific preparation method of the tetrazine dicarboxamide liposome in this example is the same as that in Example 2, and the difference only lies in the raw materials and dosages used in this example, which will not be repeated here. A slightly milky white drug liposome solution was prepared.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 246.9 nm, a polydispersity coefficient of 0.305, and a drug encapsulation rate of 70.7%.

Example 7:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 8 mg;

Cholesterol: 16mg;

Phosphatidylinositol: 160mg;

Vitamin E: 3mg;

Dichloromethane: 18mL;

Phosphate buffer solution (pH 7.2): 32 mL.

The specific preparation method of the tetrazine dicarboxamide liposome in this embodiment is the same as the method in Embodiment 3, and the difference only lies in the use of raw materials and dosages used in this embodiment, which will not be repeated here. A slightly milky white drug liposome solution was prepared.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 264.7 nm, a polydispersity coefficient of 0.255, and a drug encapsulation rate of 69.7%.

Example 8:

Tetrazine dicarboxamide liposome preparation, the liposome includes the following components and quality:

Tetrazine dicarboxamide: 3 mg;

Cholesterol: 22mg;

Lecithin (90% PC content): 90mg;

Vitamin E: 2mg;

Chloroform: 12mL;

Phosphate buffer solution (pH 6.4): 26 mL.

The specific preparation method of the tetrazine dicarboxamide liposome in this example is the same as that in Example 4, and the difference only lies in the raw materials and dosages used in this example, which will not be repeated here. A slightly milky white drug liposome solution was prepared.

The obtained tetrazine dicarboxamide liposome solution had a particle size of 327.5 nm, a polydispersity coefficient of 0.169, and a drug encapsulation rate of 71.5%.

The tetrazine dicarboxamide liposome solution obtained in the above embodiment was selected, appropriately diluted with water for injection, and the particle size of the particles in the solution was tested by a laser nanometer. The specific test results are shown in Figure 2 to Figure 7. It can be seen from the particle size distribution diagram that the particle size of the prepared tetrazine dicarboxamide liposome solution is at the nanometer level, and the solution is slightly clear and milky white. Through the implementation of the present invention, the solubility of tetrazine dicarboxamide is enhanced, which is beneficial to the exertion of its medicinal effect.

Fig. 8 is the scanning electron microscope image of the tetrazine dicarboxamide liposome solution obtained in Example 3, the solution was diluted 25 times, stained with 2% sodium phosphotungstate solution, a small amount was dropped on a silicon wafer, and the temperature was at room temperature. After volatilization and drying, the liposome morphology was observed under a scanning electron microscope. It can be seen from the electron microscope that it was spherical.

Get the tetrazine dicarboxamide liposome solution obtained in embodiment 4, use zeta potential measuring instrument to carry out zeta potential test, after testing, the zeta potential of tetrazine dicarboxamide liposome solution of the present invention is-63.97mV, It can be seen from the results that the solution is relatively stable.

The above are only preferred specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any person skilled in the art can obviously obtain the simplicity of the technical solution within the technical scope disclosed in the present invention. Variations or equivalent substitutions fall within the protection scope of the present invention.

Claims (12)

1. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 4 mg; lecithin: 100 mg; cholesterol: 20 mg; ethanol: 10 mL; phosphate buffer solution: 20mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 6.8.

2. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 4 mg; lecithin: 150 mg; cholesterol: 15 mg; vitamin E: 2 mg; diethyl ether: 15 mL; phosphate buffer solution: 25mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 6.4.

3. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 8 mg; bean lecithin: 80 mg; cholesterol: 10 mg; vitamin E: 6 mg; chloroform: 25 mL; phosphate buffer solution: 12mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 7.0.

4. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 6 mg; sphingomyelin: 200 mg; cholesterol: 25 mg; vitamin E: 8 mg; dichloromethane: 20 mL; phosphate buffer solution: 40mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 7.6.

5. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 5 mg; phosphatidylserine: 50 mg; cholesterol: 12 mg; diethyl ether: 30 mL; phosphate buffer solution: 44mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 7.2.

6. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 6.5 mg; phosphatidyl glycerol: 250 mg; cholesterol: 22 mg; ethanol: 28 mL; vitamin E: 5 mg; phosphate buffer solution: 30mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 6.8.

7. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 8 mg; phosphatidylinositol: 160 mg; cholesterol: 16 mg; dichloromethane: 18 mL; vitamin E: 3 mg; phosphate buffer solution: 32mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 7.2.

8. A tetrazine dicarboxamide liposome preparation is characterized by being prepared from the following components:

tetrazine dicarboxamide: 3 mg; lecithin: 90 mg; cholesterol: 22 mg; vitamin E: 2 mg; chloroform: 12 mL; phosphate buffer solution: 26mL, wherein the phosphate buffer solution is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate and water for injection, and the pH value of the phosphate buffer solution is 6.4.

9. A method of preparing the liposome formulation of tetrazine dicarboxamide of any of claims 2-4 and 6-8, comprising the steps of:

(1) adding tetrazine diformamide, phospholipid material, cholesterol and vitamin E into an organic solvent, stirring or carrying out ultrasonic treatment to completely dissolve the tetrazine diformamide, the phospholipid material, the cholesterol and the vitamin E to obtain a solution A;

(2) preparing by a thin film dispersion method: placing the solution A in a flask, removing the organic solvent by rotary evaporation to form a dry film on the wall of the flask,

adding phosphate buffer solution into a flask, carrying out ultrasonic hydration by a probe, and filtering by a 0.45um microporous filter membrane to prepare liposome solution; or the like, or, alternatively,

(3) preparing by an injection method: and injecting the solution A into a phosphate buffer solution which is continuously stirred through a needle cylinder to obtain a mixed solution, placing the mixed solution into a constant-temperature water bath at 50-60 ℃, continuously stirring, volatilizing to remove the organic solvent, and filtering through a 0.45-micrometer microporous filter membrane to obtain a liposome solution.

10. A method of preparing the tetrazine dicarboxamide liposomal formulation of claim 1 or 5, comprising the steps of:

(1) adding tetrazine diformamide, phospholipid material and cholesterol into an organic solvent, stirring or carrying out ultrasonic treatment to completely dissolve the tetrazine diformamide, the phospholipid material and the cholesterol to obtain a solution A;

(2) preparing by a thin film dispersion method: placing the solution A in a flask, removing the organic solvent by rotary evaporation to form a dry film on the wall of the flask,

adding phosphate buffer solution into a flask, carrying out ultrasonic hydration by a probe, and filtering by a 0.45um microporous filter membrane to prepare liposome solution; or the like, or, alternatively,

(3) preparing by an injection method: and injecting the solution A into a phosphate buffer solution which is continuously stirred through a needle cylinder to obtain a mixed solution, placing the mixed solution into a constant-temperature water bath at 50-60 ℃, continuously stirring, volatilizing to remove the organic solvent, and filtering through a 0.45-micrometer microporous filter membrane to obtain a liposome solution.

11. The method for preparing the tetrazine dicarboxamide liposome preparation according to claim 9, wherein the prepared liposome solution has a particle size of less than 400nm, a polydispersity of less than 0.4, and a drug encapsulation efficiency of more than 60%.

12. The method for preparing the tetrazine dicarboxamide liposome preparation according to claim 10, wherein the prepared liposome solution has a particle size of less than 400nm, a polydispersity of less than 0.4, and a drug encapsulation efficiency of more than 60%.

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