EA022182B1 - Method of producing docetaxel liposome form - Google Patents

Abstract

The invention relates to pharmaceutics. A method comprises preparing a composition of phosphatidylcholine and docetaxel, drying the mixture, emulsification thereof in aqueous solution, homogenation, adding a cryoprotector, sterilyzing filtration and cool dehumidification. The obtained result provides in standard and stability of the prepared liposomes (their size is presented in a narrow range) comprising docetaxel, high encapsulation of docetaxel in liposomes, docetaxel stability in a storage shelf life and reduction of toxic effect.

Description

The invention relates to pharmaceuticals.

It is well known to use taxanes [3, 5, 14], in particular, docetaxel, as an antitumor agent for treating cancer patients: locally progressive or metastatic breast cancer, non-small cell lung cancer, metastatic ovarian carcinoma, metastatic prostate cancer. Docetaxel is a semi-synthetic substance with the chemical formula C ₄₃ H ₅₃ NO ₁₄ , which is obtained from the biomass of European yew needles. Docetaxel has a unique mechanism of action and a wide spectrum of anticancer activity. The drug contributes to the accumulation of tubulin in the microtubules of cells and prevents their decay, which leads to disruption of mitosis and interfacial processes in tumor cells. In addition, docetaxel is active against certain cells that produce an excessive amount of p-glycoprotein, which is encoded by the multiple resistance gene. The use of docetaxel in advanced cancer in the 2nd line of chemotherapy is quite effective in most solid tumors [5, 11, 14].

At the same time, docetaxel is characterized by high toxicity, which manifests itself in a number of side effects: hematological reactions, fluid retention, reactions from the digestive tract, nervous and cardiovascular systems, liver, etc. According to a number of authors, patients who use docetaxel are more likely to neutropenia is noted and they have to be hospitalized and granulocytic colony stimulating factor is used. In addition, docetaxel is characterized by extremely low solubility in water, which complicates the selection of suitable dosage forms and significant technological difficulties in production. Currently, docetaxel is made and administered on a carrier containing tween-80 (polysorbate) and dissolved in 13.0% ethanol. The stability of docetaxel dissolved in ethanol is quite low and the drug is destroyed in alcohol within 4 hours. It should also be noted that Tween-80 is a complex compound with a difficult to determine molecular structure.

One of the priority areas for the development of modern pharmaceutical technologies is the development and creation of therapeutic systems of directed action. To solve this problem, nanosomal drug carriers are successfully used that can increase the focus of action and provide an increase in bioavailability. In recent years, there have been reports of the inclusion of taxanes in nanoparticles from various polymer carriers, pegylated particles and liposomes from natural and synthetic lipids [6, 7, 15, 16].

The literature describes many attempts to overcome drugs with low solubility formulations of pharmaceutical substances in aqueous solvents. Unfortunately, such formulations are developed mainly at the empirical level and there is a lack of systematic quantitative studies of how these formulations can make drug substances soluble. We previously proposed drugs based on liposome compositions, which include hydrophobic drugs: Lipoflavon, which is a phosphatidylcholine drug with bioflavonoid encapsulated in them - quercetin, used as an injection in cardiology and as eye drops; The lyoliv, in which the hepatoprotector is an antral and other medicinal hydrophobic substances in liposomes [1, 12, 13]. Clinical studies of liposome preparations based on hydrophobic and hydrophilic substances have demonstrated high pharmacological activity in pulmonology, oncology, cardiology, nephrology, ophthalmology [5, 6, 9]. Moreover, the use of liposomal forms of cytostatics (anthracycline antibiotics) [2] is a promising direction for overcoming drug resistance to chemotherapy drugs, as well as reducing the toxic effect of free forms of antitumor drugs on organs and tissues [8-10].

A known method of producing a liposome composition based on natural phosphatidylcholine and docetaxel, which has antitumor activity [15]. The method of obtaining the liposomal composition, which is carried out in accordance with [15], is selected by the prototype of the claimed method object as its closest analogue in terms of the sum of features, namely, the essence and sequence of the basic operations and techniques, the chemical nature of phosphofolipid and docetaxel, the liposomal organization of the target product the proximity of its pharmacotherapeutic manifestations.

The known method [15] involves mixing the phospholipid component in the aqueous phase with docetaxel (substance) or in an organic solvent in the presence of sodium oleate, suspending at ambient temperature, dispersing through Myugoyshbyuk at 15,000 ρδί, adding a cryoprotectant (trehalose) to liposomes to a final concentration of 10% ; the use of the resulting preparation both in liquid form and after lyophilization. Liposomes had a size before lyophilization of not more than 100 nm.

However, the method of obtaining the prototype involves operations and methods that impede its implementation and contribute to lowering the quality of the liposomal product. Firstly, this is due to the use of soybean phosphatidylcholine, which is a more oxidized product compared to egg yolk phosphatidylcholine (soybean phosphatidylcholine oxidation index is 0.35; egg phosphatidylcholine oxidation index is not more than 0.21). Secondly, the introduction of homogenization into

- 1 022182 an emulsion of docetaxel in powder form (substance) without taking into account its dissolution impairs the stability of the system and can alter the resulting system of liposomes. Thirdly, in the method [15] there is no buffer system stabilizing docetaxel in an aqueous solution. In addition, the proposed [15] system for adding trehalose to liposomes does not allow the cryoprotector to be located both inside the liposome and in the surrounding water, which leads to product instability after lyophilization.

All this reduces the effectiveness of the prototype method in the process of reproduction of the drug, as well as its stability as the target product as a chemotherapeutic agent.

The objective of the claimed method is to simplify the process of obtaining and improving the quality of the liposomal preparation in terms of stability of both the liposomal structure and the stability of the pharmacologically active substance of docetaxel, while the result obtained in solving this problem is to achieve standardization and stability of the obtained liposomes (the size of liposomes is presented in narrow range) containing docetaxel, high encapsulation of docetaxel in liposomes, stability of docetaxel during storage and underreporting toxic effect of the liposomal docetaxel (in particular, more than three times compared to neliposomalnym analogue).

To achieve the result, a method for producing a liposomal antitumor drug is proposed, which includes creating a composition of phosphatidylcholine and docetaxel, drying the mixture, emulsifying it in an aqueous medium, dispersing the emulsion, homogenizing, adding a cryoprotectant, sterilizing filtration and freeze drying, while emulsifying is carried out in an aqueous medium containing citric acid in a concentration (0.33-0.39 mg / ml), homogenization was carried out in two stages: initial at 500 atm, subsequent at 8 00 atm, the addition of a cryoprotectant - lactose was carried out fractionally: initially during emulsification, and then during homogenization, while the ratio of the components in the resulting preparation of docetaxel: phosphatidylcholine: the cryoprotectant is in the range 1: (18.0-25.0) :( 22- 63) respectively.

In general terms, in the process of practical implementation, the method involves the use of egg phosphatidylcholine and docetaxel in organic solvents, removing the solvent by evaporation in vacuo, dispersing the resulting mass in an aqueous medium with a pH of 5.4-5.8, dispersing multilayer liposomes with increasing pressure, adding a cryoprotectant both at the beginning of homogenization, and when the liposome size reaches 150-200 nm, obtaining liposomes of 80-140 nm in size, sterilizing the filtration of the obtained liposome emulsion, spilling into bottles lyophilization and sealing in an atmosphere of inert gas (or nitrogen).

The method is illustrated by graphs of the pharmacokinetic profiles of docetaxel in the liver, brain, kidneys and heart of rats (Fig. 1-4, respectively).

The effectiveness of the proposed method for the qualitative and quantitative identification of the finished product of the lipid component - phosphatidylcholine was evaluated by thin-layer chromatography on plates by chromatogram of a solution of the target product in methanol, on which the main yellow spot is at the level of the main spot of a solution of a standard sample of phosphatidylcholine;

the lipid oxidation index was carried out by UV spectroscopy at two wavelengths: 233 and 215 nm.

The effectiveness of the proposed method for qualitative and quantitative identification in the finished product of docetaxel was evaluated by high performance liquid chromatography, which was carried out to quantify foreign impurities and the content of docetaxel in the samples of the drug. Used a Ztab chromatograph /, a chromatographic column with a size of 240x4.6 mm, C18 with a particle size of 5 μm, mobile phases: mobile phase A — 0.35 g / l ammonium acetate pH 5.5; mobile phase B - acetonitrile; the speed of the mobile phase is 1.6 ml / min; detection at a wavelength of 232 nm; column temperature 30 ° C.

The size of liposome particles was determined on a nanosizer ΖοΚιΧζογ Ναηο Ζδ, Layout using photon correlation spectroscopy. Particle size was measured using a semiconductor laser at a wavelength of 375 nm.

The following examples illustrate the method for producing the liposomal preparation of Docetaxel, as well as production options. It is intended that although preferred embodiments of the invention are disclosed in the detailed description and specific examples, they are provided for illustrative purposes only, as various changes and improvements will be apparent to those skilled in the art from the description as well as the claims, without departing from the spirit and scope. inventions.

Example 1

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 3420 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel, 230 ml of buffer mixture with pH 5.62 was added, containing 1.5 g of lactose (from the required 4.18 g) and 87.5 mg of citric acid at a temperature of 40 -43 ° C. The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.821 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.8%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. The initial pressure (only two cycles) was 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (2.78 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration is 0.814 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration of 148.4 nm is 92.1%, 27.78 - 7.9%. The concentration after sterilizing filtration is 0.793 mg / ml. Inclusion after sterilizing filtration is 97.4%. The volume of the sterile emulsion is 200 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 140.0 nm - 94.6%, 38.5 nm - 5.4%; pH 5.65. The inclusion of at least 96.5%. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose (1:18:22).

Example 2

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 5.66, which contained 2.5 g of lactose (of the required 6.85 g) and 80.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.827 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.65%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only two cycles) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (4.35 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration is 0.812 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration 138.0 nm - 94.6%, 54.3 - 5.4%. Concentration after sterilizing filtration is 0.799 mg / ml. Inclusion after sterilizing filtration - 98.5%. The volume of the sterile emulsion is 202 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is kept in the nanoscale - 146.0 nm - 92.6%, 54.5 nm - 7.4%; pH 5.65. The inclusion of at least 96.5% of docetaxel. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 36).

Example 3

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4750 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel, 230 ml of a buffer mixture with a pH of 5.60 was added, in which there were 5.0 g of lactose (from the required 11.97 g) and 75.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.817 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.65%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only two cycles) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (6.97 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration is 0.814 mg / ml. Emulsion

- 3 022182 was filtered through a cascade of filters with a final filter of 0.2 μm. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration of 141.4 nm is 90.1%, 43.6 - 9.9%. Concentration after sterilizing filtration is 0.800 mg / ml. Inclusion after sterilizing filtration is 97.0%. The volume of the sterile emulsion is 200 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 153.0 nm - 90.6%, 68.5 nm - 9.4%; pH is 5.72. The inclusion of at least 96.5%. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1:25:63).

Example 4

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 4.8, which contained 5.0 g of lactose (from the required 11.97 g) and 100.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.824 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.65%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. The initial pressure during the first two cycles (four cycles in total) is 500 atm. Then homogenization was continued for two cycles at 800 atm to a particle size of not more than 180 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (2.42 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. In this example, the number of homogenization cycles was two more than in examples 1-3. The concentration of docetaxel before sterilizing filtration is 0.818 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration - 147.4 nm - 91.2%, 69.78 - 8.8%. The concentration after sterilizing filtration is 0.756 mg / ml. Inclusion after sterilizing filtration is 92.42%. The volume of the sterile emulsion is 200 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 140.0 nm - 94.6%, 38.5 nm - 5.4%; pH 4.95. The inclusion of at least 96.5%. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 63).

Example 5

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 6.56, which contained 2.5 g of lactose (of the required 6.85 g) and 67.5 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.821 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.5%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only two cycles) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 180 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (4.35 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration 0.804 mg / ml. The emulsion was filtered through a cascade of filters, with a 0.2 micron finish filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration of 148.4 nm is 92.1%, 27.78 - 7.9%. The concentration after sterilizing filtration is 0.798 mg / ml. Inclusion after sterilizing filtration - 99.25%. The volume of the sterile emulsion is 206 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization, it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor. The particle size is stored in the nanoscale range - 140.0 nm - 94.6%, 38.5 nm - 5.4%; pH 6.75. The inclusion of at least 96.5%. Storage

- 4 022182 preparations at a temperature of 2 to 8 ° C for 6 months showed the appearance of additional impurities of docetaxel up to 2.4%, which indicates the instability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 36).

Example 6

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 5.66, which contained 2.5 g of lactose (of the required 6.85 g of lactose) and 80.0 mg of citric acid at a temperature of 40-43 ° FROM. The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.827 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.65%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only one cycle) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (4.35 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration is 0.817 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration 158.0 nm - 84.6%, 95.3 nm - 10.2%, 34.3 - 5.2%. Thus, when using only 1 cycle at 500 atm, liposomes heterogeneous in size are formed. Concentration after sterilizing filtration 0.756 mg / ml. Inclusion after sterilizing filtration is 92.53%. The volume of the sterile emulsion is 200 ml. The emulsion is poured into bottles, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 166.0 nm - 90.6%, 84.5 nm - 6.4%, 45.0 nm - 3.0% pH - 5.75. Inclusion after lyophilization of 88.5% docetaxel. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. Summing up, when using 1 cycle of homogenization at 500 atm after lyophilization, nanosizes were preserved, but they were represented by heterogeneous size liposomes. In this example, a decrease in the inclusion of docetaxel in liposomes was detected. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 36).

Example 7

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 5.66, which contained 2.5 g of lactose (of the required 6.85 g) and 80.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.827 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.7%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only three cycles) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (4.35 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel before sterilizing filtration is 0.812 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration 138.0 nm - 94.6%, 54.3 - 5.4%. Concentration after sterilizing filtration is 0.799 mg / ml. Inclusion after sterilizing filtration - 98.5%. The volume of the sterile emulsion is 202 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is kept in the nanoscale - 146.0 nm - 92.6%, 54.5 nm - 7.4%; pH 5.65. The inclusion of at least 96.5% of docetaxel. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 36).

Example 8. Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine egg yolk in an amount of 2280 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary

- 5 022182 evaporator at 42-45 ° C for 120 minutes Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel, 230 ml of a buffer mixture with a pH of 5.60 was added, in which there were 5.0 g of lactose (from the required 11.97 g) and 75.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.817 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.6%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initially, the pressure (only two cycles) is 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 200 nm. Upon reaching the specified particle size, the remaining sterile lactose solution (6.97 g) was added to the emulsion. Homogenization was carried out to a particle size of not more than 150 nm. The concentration of docetaxel to a sterilizing filtration of 0.814 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration of 141.4 nm is 90.1%, 43.6 - 9.9%. The concentration after sterilizing filtration is 0.712 mg / ml. Inclusion after sterilizing filtration - 87.46%. The volume of the sterile emulsion is 200 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 153.0 nm - 90.6%, 68.5 nm - 9.4%; pH is 5.72. The inclusion of at least 96.5%. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1:12:63). As can be seen from this example, a decrease in the amount of phosphatidylcholine led to a decrease in the inclusion of docetaxel in liposomes.

Example 9

According to the prototype method [15]. 500 mg of docetaxel, 6 mg of sodium oleate and 6 g of soybean phosphatidylcholine were dissolved in chloroform, concentrated in vacuo on a rotary evaporator at 42-45 ° С for 120 min until chloroform was completely removed.

The resulting mass was dispersed in 100 ml of water using 200 rpm magnetic stirrers for 10 minutes at ambient temperature. Dispersion of multilayer liposomes was carried out through Myugoyshbyuk at 15,000 ρδί. Conducted 3 cycles of homogenization and obtained liposomes with a size of 40-60 nm in diameter. Powder trehalose was added to the liposomes to a final concentration of 10%. The emulsion was filtered through a cascade of filters with a 0.2 micron filter.

Filtration was slow: 100 ml was filtered for at least 15 minutes. The particle size after sterilizing filtration of 178.4 nm is 90.1%, 56.23 - 9.9%. Concentration after sterilizing filtration 4.7 mg / ml. The amount of impurities in docetaxel is 1.62%. Inclusion after sterilizing filtration 94.0%. The volume of the sterile emulsion is 96 ml. The emulsion is poured into bottles, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 160.0 nm - 94.6%, 48.5 nm - 5.4%; pH 6.85. The inclusion of at least 92.5%. Storage of the drug at a temperature of 2 to 8 ° C for 6 months showed the appearance of additional impurities of docetaxel up to 3.7%, which indicates the instability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: cryoprotectant (1:12:20).

Example 10

Docetaxel trihydrate in an amount of 190 mg was dissolved in 6.5 ml of chloroform, phosphatidylcholine of egg yolk in an amount of 4085 mg was dissolved in 100 ml of chloroform. Chloroform solutions were combined, filtered through 0.22 μm membranes and concentrated in vacuo on a rotary evaporator at 4245 ° С for 120 min. Then, the obtained lipid film was treated with nitrogen gas with stirring (60-80 rpm) for 1 h until complete removal of chloroform.

To a lipid film containing phosphatidylcholine and docetaxel was added 230 ml of a buffer mixture with a pH of 5.66, which contained 6.85 g of lactose (of the required 6.85 g) and 80.0 mg of citric acid at a temperature of 40-43 ° C . The container with the emulsion was stirred at 130-150 rpm for 2 hours, maintaining a temperature of 40-43 ° C. Emulsion control: 0.827 mg / ml docetaxel concentration. The amount of impurities at the level of the initial substance of docetaxel did not exceed 1.65%.

The emulsion was transferred to a high pressure homogenizer and homogenized at a temperature of 38-43 ° C. Initial pressure (only two cycles) 500 atm. Then homogenization was continued at 800 atm to a particle size of not more than 150 nm. The concentration of docetaxel to a sterilizing filtration of 0.812 mg / ml. The emulsion was filtered through a cascade of filters with a 0.2 micron filter. Filtration was satisfactory: 230 ml was filtered for no more than 10 minutes. The particle size after sterilizing filtration 138.0 nm - 94.6%, 54.3 - 5.4%. Concentration after sterilizing filtration 0.723 mg / ml. Inclusion after sterilizing filtration - 89.0%. The volume of the sterile emulsion - 205

- 6 022182 ml. The emulsion was poured into vials, lyophilized and sealed in an atmosphere of inert gas (nitrogen). According to the results of the control of the product obtained by the proposed method, after lyophilization it was found that the drug is presented in the form of a light amorphous mass of white with a characteristic odor; the particle size is stored in the nanoscale range - 136.0 nm - 93%, 64.5 nm - 7.4%; pH 5.65. Inclusion of at least 88.0% docetaxel. Storage of the drug at a temperature of 2 to 8 ° C for 6 months demonstrated the stability of the product. The ratio of components in the preparation: docetaxel: phosphatidylcholine: lactose - (1: 21.5: 36). As can be seen from this example, the addition of cryoprotectant before homogenization led to a decrease in the inclusion of docetaxel in liposomes.

Example 11

In a study of the pharmacokinetics of liposomal and non-liposomal docetaxel with a single injection of rats, it was rapidly excreted from blood plasma with a maximum concentration of about 500-1500 ng / ml. Plasma concentrations of liposomal and non-liposomal docetaxel did not differ.

Significant differences were observed in the determination of docetaxel in rat liver. Stach for the liposomal form was about 25 μg / g (T _max - 0.25 h), for non-liposomal form - about 70 μg / g (T _max 8 h). Liposomal docetaxel was almost completely eliminated from the liver by 72 h (76 ng / g), in contrast to the non-liposomal docetaxel, which persists in the liver by 72 h after administration at 56 μg / g, with a tendency to its cumulation. The pharmacokinetic profiles of docetaxel in rat liver are shown in FIG. one.

Differences were also observed for the brain - liposomal docetaxel practically did not penetrate the BBB and was detected in small doses (up to 1500 ng / g) no later than 1.5 hours after administration. Non-liposomal docetaxel was detected in rat brain even 72 hours after administration in the concentration range of 530-2330 ng / g. The pharmacokinetic profiles of docetaxel in rat brain are shown in FIG. 2.

Similar pharmacokinetic profiles were observed for the kidneys (Fig. 3): for liposomal docetaxel, C _max was about 38 μg / g, for non-liposomal, 30 μg / g (T _max 0.25 h in both cases). Moreover, in contrast to the non-liposomal form, the pharmacokinetic profile of the liposomal docetaxel in the kidneys showed a second maximum (about 15 μg / g) 24 hours after administration.

Similar to that for the kidneys, a picture was observed for the heart (Fig. 4). For liposomal docetaxel, C _max was about 18 μg / g, for non-liposomal docetaxel - 11 μg / g (T _max 0.25 h in both cases). Moreover, in contrast to the non-liposomal form, the pharmacokinetic profile of the liposomal docetaxel in the kidneys showed a second maximum (about 3.5 μg / g) 24 hours after administration.

The low penetration of liposomal docetaxel into the liver and brain leads to the conclusion about its potentially low hepatotoxicity and neurotoxicity.

Summing up, the obtained research results indicate that the product obtained according to the claimed method is homogenization in two cycles: initially at 500 atm and subsequent at 800 atm to the specified size; use for stabilization of docetaxel citric acid (0.33-0.39 mg / ml); and the ratio of components in the final preparation: docetaxel: phosphatidylcholine: lactose - 1: 18.0-25.0: 22.0: 63.0 (see examples 1-4), ensure the stability of the liposome composition with docetaxel included in them and have low hepatotoxicity and neurotoxicity. Reproduction of the claimed method when changing parameters (see examples 5-9), as well as the method according to the prototype (example 10) entails difficulties in implementing the method itself and reducing the quality of the target product, namely an increase in the size and heterogeneity of the dispersion composition of liposomes (example 6); the instability of docetaxel in the composition of liposomes, which is accompanied by an increase in impurities (example and 10);

a decrease in the inclusion of docetaxel in liposomes and instability of liposome sizes (examples 6, 8, 9, 10), a significant increase in the filtration time of the liposome emulsion (almost 4 times) (example 10).

The method of high performance liquid chromatography was used to study the stability of docetaxel after lyophilization of liposome samples compared to the original substance. The identity of the qualitative and quantitative composition of docetaxel after drying and subsequent rehydration of liposome samples was confirmed. The content of impurities and their retention time have not changed, which may indicate the stability of docetaxel in the process of obtaining liposomes and their subsequent freeze-drying.

Thus, the use of nanoparticles, which are artificial membranes - liposomes loaded with a hydrophobic cytostatic docetaxel, are of undoubted interest for further study. In addition, the introduction into practice of a drug having a dual antitumor and membrane-protective effect allows improving the quality of treatment by reducing toxic effects.

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Claims (1)

A method for producing a liposomal antitumor preparation, comprising mixing solutions of phosphatidylcholine and docetaxel in chloroform, drying the mixture, emulsifying it in an aqueous medium, dispersing an emulsion, homogenizing, adding a cryoprotectant, sterilizing filtration and lyophilizing drying, characterized in that the emulsification is carried out in an aqueous medium, which comprises emulsification in an aqueous medium containing lemon acid in a concentration (0.33-0.39 mg / ml), homogenization is carried out in at least two stages: initial at 500 atm, subsequent at 800 atm, added cryoprotectant - lactose is carried out fractionally: initially during emulsification, and then during homogenization, while the mass ratio of components in the resulting docetaxel: phosphatidylcholine: cryoprotector is in the range 1: (18.0-25.0) :( 22-63) respectively.