RU2746320C1 - Method for producing lipid mixture and liposomal oral antiviral therapeutic and prophylactic agent using said lipid mixture - Google Patents

Abstract

FIELD: lipids; pharmaceuticals.

SUBSTANCE: present invention relates to a method for producing a lipid mixture, comprising preparing an alcoholic solution of phosphatidylcholine and a solution of alpha-tocopherol and cholesterol in chloroform, mixing these solutions and removing chloroform under vacuum, while to remove alcohol and chloroform residues from the lipid mixture, said mixture is lyophilized in the presence of purified water placed in open containers separately from the alcohol solution of the lipid mixture on the shelves of the sublimation chamber, and the ratio of the volume of alcohol to the volume of water is at least 1 : 2, with the receipt after the end of the lyophilization process of a solid lipid mixture with a residual amount of organic solvents: chloroform - less 0.5 ppm., Ethanol - less than 0.3 ppm. The present invention also relates to a liposomal oral antiviral prophylactic agent.

EFFECT: technical result of the proposed invention is to increase the shelf life of the liposomal dosage form, in which the activity of alpha-interferon remains at a constant level for up to 42 months.

2 cl, 4 ex, 1 tbl, 1 dwg

Description

The invention relates to medicine, pharmaceutical industry, in particular to antiviral drugs, and concerns the creation of liposomal dosage forms containing interferon.

In clinical practice, for the treatment of viral infections, in particular hepatitis B, recombinant alpha-2-interferon, one of the known subtypes of leukocyte interferon, is widely used.

It is known that the confinement of recombinant alpha-interferon in liposomes enhances its antiproliferative activity in an experiment on tumor cells (Eppstein DA, Stewart WE Altered Pharmacological Properties of Liposome-Associated Human Interferon-Alpha. J. Virology, 1982, vol. 41, No. 2, p. 575-582). Moreover, various methods of association with the liposome membrane, depending on the lipid composition, lead to altered pharmacokinetics of interferon drugs in an experiment on mice (Killion JJ, Fan D., Bucana CD, Frangos DN, Price JE, Fidler IJ Augmentation of Antiproliferative Activity of Interferon Alfa Against Human Bladder Tumor Cell Lines by Encapsulation of Interferon Alta Within Liposomes. J. of the Nat. Cancer Inst, 1989, vol. 81, No. 18, p. 1387-1392).

Known antiviral drug for local action for intradermal administration based on interferon-alpha, enclosed in liposomes (international application, WO No. 91/01719, IPC A61K 9/127, publ. 1991). The drug was studied on an experimental model of herpes infection in guinea pigs. Liposomes are derived from lipids characteristic of the stratum corneum.

Known liposomal dosage form for oral administration, containing human alpha-interferon (US patent No. 5503828, IPC A61K 38/00, publ. 02.04.1996). However, the specific composition of the dosage form is not indicated.

Known another antiviral drug based on recombinant interferon beta, enclosed in liposomes, intended for local or parenteral (subcutaneous, intramuscular) use (European patent No. 172007, IPC A61K 9/127, publ. 1986). For parenteral administration, the antiviral drug in liposomal form contains:

Recombinant interferon beta, ME 2 × 10 ⁸ Human serum albumin, g 1.25 Dextrose, g 1.25 A mixture of diarachidoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (7: 3), mmol 2.0 Saline solution (0.9%), ml 100

The disadvantage of such a liposomal preparation is the content of human serum albumin in the composition, which leads to the possible development of allergic conditions in patients who use interferon for a long time. In addition, the method of obtaining this composition provides for the preliminary dissolution of lipids that form a liposome in organic solvents, such as propanol, butanol, cyclohexane. Despite the subsequent lyophilization of the lipid mixture, trace amounts of these solvents may be present in the final drug composition, causing unwanted side effects in patients using the drug. In addition, the composition of this composition does not provide the possibility of obtaining a freeze-dried biologically active drug suitable for long-term storage, which significantly reduces the possibility of using the drug for medical purposes.

Known liposomal antiviral drug for oral administration, containing a biologically active component - recombinant interferon-alpha-2, components that form liposomes - phosphatidylcholine and cholesterol, stabilizer - sucrose, antioxidants - alpha-tocopherol and ascorbic acid, which is a lyophilisate or RF patent No. 2123328, IPC A61K 9/127, publ. 20.12.98), with the following ratio of lyophilisate components, mg:

Recombinant interferon-alpha-2, ME (2.5-10) × 10 ⁵ Phosphatidylcholine 60-80 Cholesterol 6-10 alpha-tocopherol 6-10 Vitamin C 1-2 Sucrose 40-90

Such a liposomal drug provides the manifestation of the biological activity inherent in interferon-alpha-2 during the oral route of administration of the drug and the achievement of a therapeutic effect from the use of this dosage form without the manifestation of side effects accompanying the parenteral route of administration of interferon.

However, the specified liposomal drug has an insufficient shelf life (the activity of interferon alpha-2 is practically at a constant level for no more than 12 months).

The closest analogue (prototype) is a liposomal oral antiviral therapeutic and prophylactic agent (RF patent No. 2361572, IPC A61K38 / 21, publ. 20.07.2009), including recombinant human interferon-alpha, phosphatidylocholine, cholesterol, alpha-tocopherol, lactose , sodium chloride, sodium phosphoric acid disubstituted 12-water and sodium phosphoric acid monosubstituted 2-water. Moreover, it is a lyophilisate with the following content of lyophilisate components, mg:

Recombinant interferon alpha, ME (2,5-10) ⋅10 ⁵ Phosphatidylcholine 40.0-42.0 Cholesterol 4.0-5.0 Alpha-Tocopherol 0.5-0.6 Lactose 90.0-92.0 Sodium chloride 7.8-8.1 Sodium phosphoric acid disubstituted 12-water 4.0-5.0 Sodium phosphoric acid monosubstituted 2-water 0.5-0.6

However, the prototype provides an insufficient shelf life of the liposomal dosage form, in which the activity of interferon-alpha remains at a constant level for no more than 30 months due to the fact that a lipophilic mixture of phosphatidylocholine, cholesterol and alpha-tocopherol for the formation of liposome shells is obtained by removing organic solvents from their solution in a vacuum rotary evaporator.

This is how lipid films are obtained from a lipophilic mixture in many liposome technologies (Liposomes, a practical approach. Ed. By RRC New, Oxford etc., IRL Press, 1990; Bangham AD, Prog. Biophis. Mol. Biol., 1968, v. 18, pp. 29-95; Liposomes in biological systems. Ed. By Gregoriadis G. and Allison A.-M .: Medicine, 1983; US Patent No. 5654, 1995; US Pat. No. 4883665, 1990; Efremenko V I. Liposomes, Stavropol, 1999).

The presence of residual amounts of organic solvents in the lipid film at the stage of obtaining the dosage form has a negative effect both on the initial target activity of the drug and decreases it during long-term storage. It is known that organic solvents intensify the processes of chemical oxidation, including lipid peroxidation (Iriskulov, BU (2019) Modern condition of the problem of lipid peroxide oxidation. Central Asian Journal of Medicine: Vol. 2019: Iss. 1, Article 7 .) and have a denaturing ability in relation to proteins (AB Belova, VV Mozhaev, AV Levashev, MV Sergeeva, K. Martinek, Yu.L. Khmelnitsky. Relationship of physical and chemical characteristics organic solvents with their denaturing ability in relation to proteins BIOCHEMISTRY (1991). Vol. 56, issue 11, pp. 1923-1945).

The technical result of the proposed invention is to increase the shelf life of the liposomal dosage form, in which the activity of alpha-interferon remains at a constant level for up to 42 months due to the use of lipid mass with a lower content of trace amounts of organic solvents in the liposomal agent.

The specified technical result is achieved by the fact that in a liposomal oral antiviral therapeutic and prophylactic agent, including recombinant human interferon-alpha, lactose, sodium chloride, sodium phosphoric acid disubstituted 12-water, sodium phosphoric acid monosubstituted 2-water and lipidyl mixture of phosphatidyl cholesterol and alpha-tocopherol for the formation of liposome shells, according to the invention, as a lipid mixture of phosphatidylocholine, cholesterol and alpha-tocopherol for the formation of liposome shells, the agent contains a mixture obtained by removing organic solvents from it by lyophilization in the presence of purified water, placed in open containers separately from the alcoholic solution of the lipophilic mixture on the shelves of the sublimation chamber, and the ratio of the volume of alcohol to the volume of water is at least 1: 2, and the residual amount of organic solvents in the resulting lipid mixture after lyophilization is: chloroform - less than 0.5 ppm, and ethanol - less than 0.3 ppm., and the content of lyophilisate components in the final product is, mg:

Recombinant interferon alpha substation 0.0001-0.050 Phosphatidylcholine 40.0-42.0 Cholesterol 4.0-5.0 Alpha-Tocopherol 0.5-0.6 Lactose 90.0-92.0 Sodium chloride 7.8-8.1 Sodium phosphoric acid disubstituted 12-water 4.0-5.0 Sodium phosphoric acid monosubstituted 2-water 0.5-0.6

The activity of the substance of recombinant interferon-alpha is (0.25-1.0) ⋅10 ⁶ IU.

The presence of cholesterol and vitamin E in the liposomal membrane in the claimed ratios ensures the relative stability of liposomes in the gastrointestinal tract to the site of lipid absorption in the body, and, accordingly, the preservation of the biological activity of interferon contained in them.

Due to the diameter of the membrane opening to 0.1 microns (during the extrusion of the liposomal product), it is possible to reduce the size of liposomes to 0.15 microns, which made it possible to keep the loading of interferon into liposomes at the same (as in the prototype) level, since the loading factor is directly proportional to the total value the surface of the lipid bilayer and is inversely proportional to the size of the liposomes.

When using interferon in an amount of less than 250 thousand IU, insufficient clinical efficacy of the claimed dosage form is revealed when taken orally, and an increase in the amount of interferon in a single dose of more than 1 million IU is impractical, since it does not lead to a further increase in the therapeutic effect of the drug and can cause unwanted side effects. effects characteristic of high doses of interferon.

Experimental studies have shown that the concentrations of phosphatidylcholine in the range of 40-42 mg / ml, cholesterol in the range of 4.0-5.0 mg / ml and tocopherol in the range of 0.5-0.6 mg / ml are optimal for the inclusion of a given amount of interferon in liposomes with an average particle size of 0.15 μm.

The lactose content in the range of 90.0-92.0 mg / ml in the lipid composition ensures stable preservation of the liposomal membrane during lyophilization, and its content of more than 92.0 mg / ml, according to the authors, does not provide additional advantages.

The introduction of such components as sodium chloride, sodium phosphoric acid disubstituted 12-water and sodium phosphoric acid monosubstituted 2-water in the claimed concentrations ensures the preservation of the activity of interferon at the stage of preparation of the dosage form.

In addition, lactose increases the stability of the finished dosage form during storage. Lactose is a non-reducing disaccharide and therefore is subject to oxidation to a much lesser extent compared to sucrose, which made it possible (compared to the prototype) to abandon the use of ascorbic acid as an antioxidant.

Vitamin E (alpha-tocopherol), present in liposomes in the stated ratios, has a sufficient antioxidant and stabilizing effect on interferon and lipid components.

An increase in the stability of interferon at the stage of preparation of the dosage form is achieved by introducing a phosphate-saline buffer into the composition of the rehydrating solution with characteristics close to those of the interferon substance, which reduces the inactivating effect of such factors as osmolality and acid-base balance.

In addition, when obtaining a solid mixture of lipids (lipid mass) for the preparation of liposomes, from their solution in organic solvents, chloroform and ethanol, the authors used the lyophilization method, which makes it possible to almost completely remove these organic solvents. It is known that organic solvents intensify the processes of chemical oxidation, including lipid peroxidation (Iriskulov, BU (2019) Modern condition of the problem of lipid peroxide oxidation. Central Asian Journal of Medicine: Vol. 2019: Iss. 1, Article 7 .; OS Fedorova, SE Olkin, VM Berdnikov The chemiluminescence mechanism in 1,10-Phenantroline oxidation during catalytic decomposition of Hydrogen Peroxide. Z.phys. Chemie, Leipzig 263 (1982) 3, S. 529-549) and have a denaturing ability in relation to proteins (A.B. Belova, V.V. Mozhaev, A.V. Levashev, M.V.Sergeeva, K. Martinek, Yu.L. Khmelnitsky The relationship of the physicochemical characteristics of organic solvents with their denaturing in relation to proteins BIOCHEMISTRY (1991), T.56, issue 11, pp. 1923-1945;

A.A. Gidulyanov The short-term effect of denaturing hydrophobic agents on the electrophoretic mobility of hemoglobin in human blood B.I. Vernadsky, ser. "Biology, chemistry", volume 27 (66). 2014. No. 3. Pp. 26-30).

The presence of residual amounts of organic solvents at the stage of obtaining the dosage form can have a negative effect both on the initial target activity of the drug and decrease it during long-term storage.

Lyophilization of lipid solutions makes it possible to significantly (to a level less than 0.5 ppm) reduce the residual amount of these organic solvents in the final product and thereby reduce their negative inactivating effect.

FIG. 1 shows a diagram of the placement of trays with a lipid solution in an organic solvent in cassettes, at the bottom of which purified water is located, before placing the cassettes on the shelves of the lyophilization unit chamber.

The following are examples of 1-3 liposomal formulations.

Example 1. Composition with a minimum content of components, mg:

The substance of recombinant interferon-alpha with activity 0.25⋅10 ⁶ ME 0.0001 Lipophilic mixture obtained by lyophilization with residual chloroform content - less than 0.5 ppm., and ethanol - less than 0.3 ppm .: Phosphatidylcholine 40.0 Cholesterol 4.0 Alpha-Tocopherol 0.5 Lactose 90.0 Sodium chloride 7.8 Sodium phosphoric acid disubstituted 12-water 4.0 Sodium phosphoric acid monosubstituted 2-water 0.5

Example 2. Composition with an average content of components, mg:

Recombinant interferon alpha substance with activity 0.5⋅10 ⁶ ME 0.001 Lipophilic mixture obtained by lyophilization with residual chloroform content - less than 0.5 ppm., and ethanol - less than 0.3 ppm .: Phosphatidylcholine 41.0 Cholesterol 4.5 Alpha-Tocopherol 0.55 Lactose 91.0 Sodium chloride 8.0 Sodium phosphoric acid disubstituted 12-water 4.5 Sodium phosphoric acid monosubstituted 2-water 0.55

Example 3. Composition with the maximum content of components, mg:

Recombinant interferon alpha substance with activity 1.0⋅10 ⁶ ME 0.050 Lipophilic mixture obtained by lyophilization with residual chloroform content - less than 0.5 ppm., and ethanol less than 0.3 ppm. Phosphatidylcholine 42.0 Cholesterol 5.0 Alpha-Tocopherol 0.6 Lactose 92.0 Sodium chloride 8.1 Sodium phosphoric acid disubstituted 12-water 5.0 Sodium phosphoric acid monosubstituted 2-water 0.6

Example 4. Technology for obtaining a liposomal form.

Liposomes of the claimed composition with an average size of 0.15 μm are obtained by extrusion of multilamellar vesicles through polycarbonate membranes with an average pore size of 0.1 μm.

In a container (V = 1 l) containing 95% ethyl alcohol in an amount of 400 ml, add a weighed portion of phosphatidylcholine (Lipoid S-100) in an amount of (357.09 ± 0.01) g, stir and incubate for 3 hours until complete dissolving lipoid. In this case, the volume of the solution increases by approximately 0.35 liters.

Tocopherol acetate (vitamin E) in an amount of (4.85 ± 0.01) g, Cholesterol (Cholesterol) in an amount of (39.28 ± 0.01) g are weighed on a scale in a polymer glass with a capacity of 200 ml, and then 100 ml of Trichloromethane is added (Chloroform). A glass with a mixture of vitamin E, Cholesterol and Chloroform is placed on a magnetic stirrer for stirring until the Cholesterol is completely dissolved, while the volume of the solution increases by 20 ml. Then bring the volume of the resulting solution with ethyl alcohol 95% to a volume of 130 ml.

A solution of Cholesterol with vitamin E in Chloroform is introduced into the Lipoid solution in alcohol and the volume of the solution is brought up to 1000 ml with ethyl alcohol 95%, mixed.

Evacuation and lyophilization of lipid solution

A container with a capacity of 1 liter with a freshly prepared lipid solution is transferred to evacuation to remove chloroform. For this, the lipid solution is poured into pre-prepared trays, loaded into the chamber of a vacuum cabinet, and a vacuum is brought up to minus 1 bar and held for 20 minutes. The bulk of chloroform (90-95%) is evaporated and downloaded within the specified time. After that, the vacuum is released and the trays are unloaded.

The removal of ethyl alcohol and chloroform residues from the lipid solution is carried out in a lyophilization unit.

Unloaded from the vacuum cabinet, trays 1 with lipid solution are placed in cassettes 2 of a larger size, at the bottom of each of which about 400 ml of purified water 3 is poured (Fig. 1). The lipid solution in the trays 1 is not mixed with purified water 3 located at the bottom of the cassettes 2. The amount of purified water added to each cassette is selected so that two volumes of water are added to one volumetric part of ethanol in the lipid solution.

Cassettes 2 are placed on the shelves of the lyophilization chamber. Before lyophilization, the solution is frozen in the installation for at least 5 hours to a temperature not higher than minus 45 ° C. When the freezing of liquids is visually completed and the temperature on the shelves of the lyophilizer reaches minus 45 ° C, the condenser temperature - minus 75 ° C, the vacuuming of the lyophilizer working chamber is started. Two hours later, after the condenser temperature drops to minus 85 ° C, the shelf cooling is turned off. The main process of lyophilization occurs with the natural supply of heat from the environment to the working chamber of the installation. When liquids evaporate (alcohol from trays 1 and water 3 from cassettes 2), a gaseous water-alcohol mixture with a percentage of ethyl alcohol of about 30-40% is formed in the working chamber of the lyophilizer, which freezes at a temperature of minus 28-35 ° C and is therefore effectively captured a condenser cooled to a temperature of minus 85 ° С (ethyl alcohol freezes at a temperature of minus 114 ° С). After 40 hours, when the temperature in the trays reaches 15-20 ° C, the shelves of the lyophilizer are heated to 30-35 ° C at a rate of 3 ° C per hour. followed by exposure under these conditions for another 6 hours. This ends the lyophilization of lipids.

After the end of the lyophilization process, to equalize the pressure, sterile dried argon is fed through the filter to the lyophilization unit and the chamber is depressurized.

The resulting lipid mass is discharged from the lyophilization unit and transferred to the hydration stage. If necessary, the lipid mass is stored in a freezer at a temperature not exceeding minus 20 ° C. Residual amounts of organic solvents in the resulting lipid mass were monitored by headspace static gas chromatographic analysis. The chloroform content is less than 0.5 ppm. (no chromatographic peak was found), ethanol - less than 0.3 ppm. (no chromatographic peak detected).

Preparation of a solution for hydration, hydration of the lipid mass,

obtaining an intermediate product

Weigh, according to the calculation, a sample of the components in the quantities given below

lactose 125.5 g sodium chloride 11.0 g sodium phosphoric acid disubstituted 12-water 6.2 g sodium phosphoric acid monosubstituted 2-water 0.7 g

The weighed portions are transferred into a container for the preparation of solutions, water for injection is added to the calculated volume. The components are stirred until complete dissolution within (10 ± 2) min at room temperature. Control the pH of the solution (should be between 6.5 and 7.5). Next, a weighed portion of the lipid mass (130.48 g) is added to the salt solution and the contents are stirred until the lipid mass is completely hydrated for 30 minutes at room temperature. The resulting liposomal suspension is injected with a calculated amount of a solution of the substance of recombinant interferon-alpha (based on the final dosage form after lyophilization of the resulting suspension, examples 1-3).

The resulting liposomal suspension with interferon is subjected to sequential forming and sterilizing filtration through polycarbonate membranes from Nuclepore (pore diameters 0.4; 0.2 and 0.1 μm) under the pressure of an inert gas (nitrogen, argon). Under aseptic conditions, 1 ml of the suspension is poured into vials, frozen at minus 40 ° C and the preparation is lyophilized until a residual moisture content of no more than 8% is reached. Immediately after drying, the vials with the drug are hermetically sealed in an inert gas atmosphere under aseptic conditions. Before use, 1.0 ml of water is added to the lyophilized preparation and shaken until a homogeneous milky-white suspension is obtained.

The average diameter of liposomes obtained by extrusion of multilamellar vesicles through polycarbonate membranes and having the claimed composition is 0.15 μm.

When analyzing the antiviral activity of the liposomal suspension, it was found that no more than 25% of interferon remains unencapsulated.

To confirm the stability of the lyophilized liposomal dosage form of recombinant alpha-2-interferon of the claimed composition, we studied the antiviral activity of the drug for 42 months. The drug was stored in a refrigerator at a temperature of 4 ° C. During the entire observation period, the degree of lipid oxidation remained unchanged. As studies have shown, during 42 months of storage, no decrease in the antiviral activity of the proposed drug was found (see table 1).

Analysis of table 1 comparing the results of measuring the specific activity of interferon-alpha in the prototype drug and manufactured samples of the claimed drug in liposomal form shows that, compared to the prototype, the shelf life of the claimed alpha-interferon drug in liposomal form increased by 40% (by 12 months).

Note (*) compliance with the requirements of regulatory documents (not less than 80% and not more than 125% of the declared specific activity).

Claims (3)

1. A method of obtaining a lipid mixture, including the preparation of an alcohol solution of phosphatidylcholine and a solution of alpha-tocopherol and cholesterol in chloroform, mixing these solutions and removing chloroform under vacuum, characterized in that to remove alcohol and chloroform residues from the lipid mixture, said mixture is lyophilized in the presence of purified water placed in open containers separately from the alcoholic solution of the lipid mixture on the shelves of the sublimation chamber, and the ratio of the volume of alcohol to the volume of water is at least 1: 2, with the receipt after the end of the lyophilization process of a solid lipid mixture with a residual amount of organic solvents: chloroform - less 0.5 ppm, ethanol less than 0.3 ppm. 2. Liposomal oral antiviral therapeutic and prophylactic agent, including the substance of recombinant human interferon-alpha, lactose, sodium chloride, sodium phosphoric acid disubstituted 12-aqueous, sodium phosphoric acid monosubstituted 2-water and a lipid mixture of phosphatidylocholine and alpha-cholesterol for the formation of liposome membranes, characterized in that as a lipid mixture of phosphatidylocholine, cholesterol and alpha-tocopherol for the formation of liposome membranes, the agent contains a mixture obtained according to claim 1, and the content of lyophilisate components in the final product is, mg: Recombinant interferon-alpha substation with activity (0.25-1.0) ⋅10 ⁶ ME 0.0001-0.050 lipid mixture: Phosphatidylcholine 40.0-42.0 Cholesterol 4.0-5.0 Alpha-Tocopherol 0.5-0.6 Lactose 90.0-92.0 Sodium chloride 7.8-8.1 Sodium phosphoric acid disubstituted 12-water 4.0-5.0 Sodium phosphoric acid monosubstituted 2-water 0.5-0.6

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