RU2514000C1 - Liposomal composition and method of its preparation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a liposomal pharmaceutical composition that performs directional transportation of physiologically active agents in order to increase the therapeutic activity of medicinal preparations, as well as ensuring high efficiency and stability of the active substance in the liposomes.

EFFECT: increasing the degree of incorporation of the medicinal preparation in the liposome structure, and increasing the shelf life of the liposomal preparation without substantial changing the degree of incorporation of the medicinal preparation.

7 cl, 2 tbl, 5 ex

Description

The claimed invention relates to biotechnology and the pharmaceutical industry and can be used to create systems, in particular, liposomal pharmaceutical compositions that transport the physiologically active substances in order to increase the therapeutic activity of drugs, as well as ensure high efficiency and stability of the active substance in liposomes .

Liposomes - closed bubbles spontaneously formed in mixtures of phospholipids with water, the wall of which consists of one or more bilayers of phospholipids (layers with a thickness of two molecules), into which other substances, for example, proteins, are embedded. Liposomes contain water or a solution (see http://en.wikipedia.org/wiki/%CB%E8%).

Liposomes are successfully used as drug carriers, because:

in chemical composition similar to natural cell membranes;

universal, which allows you to transfer a wide range of medical chemicals;

do not cause allergic reactions.

However, there are a number of difficulties in using liposomes in medicine. First, liposomes are absorbed by the cells of the reticuloendothelial system, with more of them found in the liver, spleen, bone marrow, lymph nodes, and blood flow. Therefore, the delivery of drugs using liposomes to other organs and parts of the body is a more difficult task. Secondly, lipoproteins, exchanging lipids with liposomes, contribute to the destruction of liposomes and the outflow of their contents. In addition, the task is to increase the shelf life of liposomes after their preparation (see Kobrinsky G. Liposomes in medicine (see http://nt.ru/nj/nz/1988/0601.htm)// Science and Life. 1988. No. 6. (Electronic version: NI. Scientific journals, 2002).

Liposomes, as drug carriers, have been widely known since the mid-60s of the last century. Over the past time, both the advantages and disadvantages of liposome-based drugs have been studied. The obvious advantages of liposomes include biocompatibility, biodegradability, and the ability to transfer compounds of any structure, including polar, nonpolar, amphiphilic, with a large and small molecular weight. In addition, in many cases, targeted drug delivery can be achieved through passive targeting or conjugation of liposomes with biomarkers.

In addition to these disadvantages of liposomes as a dosage form, there are difficulties associated with the high rate of absorption of liposomes by macrophages, which leads to a short duration of the therapeutic effect of the drug. There are problems with the standardization of liposomal forms.

Currently, several different technologies are known from the prior art that make it possible to achieve a high degree of incorporation of medicinal substances into the structure of liposomes and ensure their reliable retention over the entire shelf life of the drug.

The first way is a chemical modification of the drug molecule itself in order to increase its lipophilicity. In this case, the modified molecule easily integrates into the liposome membrane, however, at the same time, it is already a completely different substance, requiring additional studies on biological activity (see Bakker-Woudenberg IA, Storm G, Woodle MC Liposomes in the treatment of infections. J Drug Target (1994) 2: 363-371.). This approach requires the implementation of a significant range of work, the full cycle of which may take a long time, from two to three years.

The second method is based on the inclusion of the drug in the internal aqueous phase of the liposomal suspension. This is achieved by double emulsifying or repeating several hydration-freezing cycles (see Hiroshi Kikuchi et al. Gene delivery using liposome technology. Journal of Controlled Release 62 (1999) 269-277).

The disadvantages of this method are due to scaling problems, because the method includes the implementation of a number of non-technological operations, including, including laboratory synthesis. In addition, when implementing this method, the drug substance can form complex compounds with the components of the membrane and due to this exit during storage of the liposomal dosage form into the external environment of the liposomal solution.

A third method is known, based on the preparation of a lipid composition having an increased affinity for a substance. Since many drugs have an increased acidity, they can easily form salts with a number of basic compounds that make up the structure of the liposome membrane, and they can be effectively deposited on positively charged liposome membranes. This approach allows for the almost complete incorporation of the drug substance into the liposome structure without the use of complex and non-technological procedures and without any modification of the drug substance itself (see Crommelin DJ Influence of lipid composition and ionic strength on the physical stability ofliposomes. J Pharm Sci (1984 ) 73: 1559-1563.).

The prior art pharmaceutical composition is a liposome emulsion formed by phospholipids in the form of liposomes with an average particle size of 0.05-0.22 microns and sulfated glycosaminoglycans, while the sulfated glycosaminoglycans contain sodium salt of keratan sulfate and sodium salt of chondroitin sulfate, and are in a certain ratio in wt.%:

Phospholipid 0.001-2 Keratan Sulfate Sodium 0.002-0.2 Sodium salt 0.006-0.1 Saline rest,

while sulfated glycosaminoglycans are located both inside the liposomes and outside the liposomes, in physiological saline (see RF patent for invention No. 2460517 “Pharmaceutical composition for the complex treatment of ocular surface diseases in patients with primary open-angle glaucoma”, filing date 07/14/2011 , published on September 10, 2012).

Known biocidal composition on a liposomal basis, containing a quaternary ammonium - didecyldimethylammonium halide in the form of a clathrate with urea and lipid (see RF patent for the invention No. 2353395 "Biocidal composition on a liposome basis", filing date 14.06.2007, published 04/27/2009 .).

Known liposomal composition containing the following ingredients, parts by weight: paclitaxel - 2-5; phosphatide - 20-200; cholesterol - 2-30; amino acids - 0.3-4; lyophilized excipient - 10-75. The method for producing this composition includes the following operations: dissolving paclitaxel, phosphatide, cholesterol in isopropanol or ethanol to obtain a clear solution, removing the solvent to form a membrane, then adding an aqueous solution of amino acids and a lyophilized excipient, followed by hydration, sonication or homogenization to obtain liposomal paclitaxel preparation (see RF patent for invention No. 2264807 “Liposome composition with paclitaxel for the treatment of cancer and sob of its receipt ”, filing date 02/28/2001, the date of publication of the application 10.09.2004).

The prior art method for producing a liposomal composition, comprising forming a mixture containing at least one component of the liposome membrane, water and an additive, dispersing the mixture reduces to the fact that either a submicro powder of a substance insoluble in water and in an organic solvent is used as an additive, either a polymer swelling in water, or a liquid not miscible with water and other components of the mixture (see RF patent for the invention No. 2104691 "Method for producing a liposomal composition (options)", date under achi 02/20/1995, the date of publication of the application 02/27/1997).

A known method for producing a liposomal composition, including the formation of a mixture of lipids with lipophilic and hydrophilic additives, fine powder and dispersion, while at the preliminary stage of preparation of the fat phase it is treated with amine-containing compounds to redistribute the charge, and partially lipids are hydrated, then they are added in the required amounts of additives, moreover, liposomes form at the interface of a two-phase system water / water-insoluble particles (see patent for the invention No. 2311449 "Method p radiation liposomal formulation ", the filing date 20.12.2005, published 27.11.2007 g).

In addition, there is a known method for producing dry liposomal preparations, comprising mixing phospholipids, a powdery carrier and a biologically active substance and further processing the resulting mixture, with the composition consisting of a biologically active substance, a phospholipid from the group consisting of vitol, being poured into the feed hopper of the jet mill and lecithin PRO, and a water-soluble powder carrier and subjected to grinding in a jet mill at a pressure of at least 2.5 bar and a gas flow rate of at least 100 l / min, with further m selection of fractions with a particle size less than 50 microns. Alternatively, a mixture of phospholipids and a powder carrier is subjected to grinding, and then a solution of the biologically active substance in a polar solvent is added to the resulting product, and the suspension is subjected to freeze drying, treating it if necessary before drying with ultrasound at a frequency of 40 kHz (see patent patent No. 2437649 "A method of obtaining dry liposomal preparations", date 03.11.2008, published 12.27.2011).

The closest technical solution to the claimed liposomal composition is a composition for the delivery of medicinal or diagnostic compounds, which is a liposome having an inner space separated from the medium by a membrane comprising one or more lipids, the inner space comprising an anion selected from a polyanionized monosaccharide or polyanionized disaccharide, and a transmembrane gradient is created on the liposome that is effective for retaining substances inside the liposome, while transmembrane gradient may be a gradient of an ammonium ion or substituted ammonium ion, as which may be used, for example, a quaternary ammonium ion, and the lipid is phosphatidylcholine or cholesterol.

A method of encapsulating a substance in a liposome involves the step of contacting the composition with the substance for a time sufficient for the substance to become encapsulated in the liposome, the portion of the substance that becomes encapsulated in the liposomes being at least 80% to 95% , and the substance is a completely cationic substance, therapeutic agent or detectable marker (see RF patent for the invention No. 2424792 "Liposomes used for drug delivery, date of filing 02.05.2005, It publishes 27.07.2011, at PCT application US 2005/15349 20,050,502).

The disadvantages of this invention are due to the impossibility of their use to obtain preparations based on strong organic acids, since in this case the formation of salts between the drug and Quaternary ammonium compounds leads to the formation of crystalline clusters on its surface, which, in turn, reduces the stability of the liposomal suspension.

The technical result to which the claimed invention is directed is to increase the degree of inclusion of the drug (active) substance in the structure of liposomes and increase the shelf life of the liposome preparation without significantly changing the degree of inclusion of the drug (active) substance.

In this case, the sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one, dihydrate (triazavirin) is used as a medicinal substance.

The specified technical result is achieved in that the liposomal pharmaceutical composition comprising a medicinal substance, lipids in the form of phosphatidylcholine and cholesterol, quaternary ammonium ions, additives, according to the invention, the sodium salt of 2-methylthio-6-nitro-1,2-4 is used as a medicinal substance -triazolo [5,1-c] -1,2,4-triazin-7-one, a dihydrate introduced into liposomes in the form of complex salts formed with quaternary ammonium salts, which are used cetylpyridinium chloride and hexadecyl trimethylammonium chloride .. Id, wherein a total content of the complex salt in an amount more than 20 wt% are used as lipids phosphatidylcholine, if they contain less than 20 wt% - phosphatidylcholine and cholesterol, wherein the components are in a certain ratio, wt.%:

2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one sodium salt, dihydrate - cetylpyridinium chloride 7.5-12.5 2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one sodium salt, dihydrate hexadecyl trimethylammonium chloride 7.5-12.5 phosphatidylcholine 75.00-80.00 cholesterol up to 5.00 additives 0.05-0.4.

Phosphatidylcholine is Epicuron 200.

Antioxidants and preservatives are used as additives, while a mixture of tocopherols (E306) in an amount of 0.05-0.1 wt.% Is used as antioxidants, and as a preservative, for example, methyl paraben in an amount of 0.1-0.3 wt. .%.

The specified result is achieved in that the method for producing a liposomal pharmaceutical composition, comprising introducing a drug into a liposome, according to the invention, a drug substance, which is used as a salt of 2-methylthio-6-nitro-1,2-4-triazolo [5,1- c] -1,2,4-triazin-7-one, dihydrate, is introduced into liposomes in the form of complex salts formed with quaternary ammonium salts, which are used cetylpyridinium chloride and hexadecyl trimethylammonium chloride, while dissolving the obtained complex salts they are removed in a non-polar solvent and mixed with lipids and additives, after which the solvent is removed, and the resulting lipid film is cooled, dried in vacuum and hydrated to form a suspension of multilamellar vesicles, from which, by ultrafiltration, carried out simultaneously with sterilization at a temperature of 45-50 ° C, form liposomes.

At the same time, phosphatidylcholine and cholesterol are used as lipids.

Active drug substance Triazavirin is introduced into the lipid composition in the form of complex salts formed with quaternary ammonium salts having surface-active and antiseptic properties. Thanks to this method of obtaining a liposome suspension, the leaching of the drug Triazavirin from the liposome structure is prevented and its lipophilicity is increased, while the complex compounds are located both inside the liposome and on its membrane, as well as in physiological saline, which makes it possible to deliver the drug most efficiently.

In this case, it is not necessary to carry out operations to modify the medicinal substance itself, which consists in introducing alkyl residues at the N-atoms of the heterocyclic nucleus and, as a result, leads to a change in the structure of the active active substance, and this, in turn, leads to radical changes in its pharmacokinetic profile . In addition, a significant set of additional studies is not required.

Triazavirin (H-form), intended for the treatment and prevention of infectious diseases of the viral nature of humans and animals, has high acidity (see patent for invention No. 2294936 "Sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [ 5,1-c] -1,2,4-triazin-7-one, a dihydrate with antiviral activity, filing date 06/29/2005, published 03/10/2007), which allows it to easily form salts with the whole a number of basic compounds and, including, to efficiently precipitate on positively charged liposome membranes, which, in turn, Allows you to achieve almost complete inclusion of Trizavirin in the structure of liposomes without any modification, without using any complex and low-tech procedures.

In the claimed invention, cetylpyridinium chloride and hexadecyl trimethylammonium chloride were used as quaternary ammonium salts, salts that crystallize well from a suitable solvent, for example, alcohol or acetonitrile, and their complexes with Triazavirin have similar physicochemical properties.

Complex salts of these compounds with triazavirin were prepared. In addition, the physicochemical properties of these salts were studied. Unlike the initial Triazavirin, salts are practically insoluble in water, but they are readily soluble, for example, in chloroform and triglycerides. Under certain conditions, salts can form continuous phases with components of the lipid films of liposomes, however, upon short standing they can crystallize from them. To overcome this drawback, a mixture of two different quaternary ammonium salts was used, which has the least tendency to crystallization, in particular, an equimolar mixture of the above cetylpyridinium chloride and hexadecyl trimethylammonium chloride was used. The use of a mixture of salts avoids crystallization of the lipid film of liposomes, which negatively affects their stability. At the same time, a positive charge on the surface of liposomes, equal to +19 mV, allows you to stabilize the surface of the lipid membrane and prevents liposome fusion during storage. Moreover, additional steric stabilization can significantly slow down the aging process of liposomes.

To prove the effectiveness of the use of the liposome composition proposed for protection, a series of experiments was carried out to introduce Triazavirin salts into the structure of liposomes. In particular, a comparison was made of liposomes obtained in accordance with the claimed invention and synthesized by direct hydration in the presence of Triazavirin. As a result, it was found that with direct hydration of the lipid film, the degree of inclusion of Triazavirin in the structure of liposomes does not exceed 35%, while when using complexes of Triazavirin with quaternary ammonium salts, it increases to 85%.

Technical solutions that coincide with the totality of the essential features of the claimed invention have not been identified, which allows us to conclude that the claimed invention meets such a patentability condition as “novelty”.

The claimed essential features that predetermine the receipt of the specified technical result, do not explicitly follow from the prior art, which allows us to conclude that the claimed invention meets such a patentability condition as "inventive step".

The patentability condition "industrial applicability" is confirmed by examples of specific applications.

The inventive liposome composition is prepared as follows.

Active ingredient Triazavirin in the form of a 5% aqueous solution (1 mmol) is added dropwise to a 5% solution of a mixture of salts of cetylpyridinium chloride (0.5 mmol) and hexadecyl trimethylammonium chloride (0.5 mmol) and stirred until a precipitate which is filtered off and then dried at a temperature of 40-50 ° C. After that, the resulting complex compound is dissolved in a 1% solution of chloroform or methanol and phosphatidylcholine, for example, of the Epicuron 200 brand, is added to it in an amount of 75% of the total solids in the solution, as well as a preservative, for which, for example, methyl paraben and an antioxidant, which is used as a mixture of tocopherols (0.05-0.1%). After that, the solvent is carefully distilled off on a rotary evaporator, getting a lipid film of liposomes, the composition of which is shown in Table No. 1.

The film is formed at an elevated temperature of 45-50 ° C, after which the composition is cooled to 0 ° C and dried in vacuum.

Table number 1 The composition of the lipid film Component The content of the component, the lower value, wt.% The content of the component, the upper value, wt.% Triazavirin 7.05 11.75 Phosphatidicholine 75 80 Cetylpyridinium chloride 4.10 6.80 Hexadecyl-trimethylammonium chloride 3.85 6.45 Cholesterol 0.1 5,0 Methylparaben (preservative) 0.1 0.3

A mixture of tocopherols (E306) (antioxidant) 0.05 0.1

Thus, the obtained liposome film contains from 15 to 25% of the complex salts of triazavirin with quaternary ammonium salts and 75-80% of the lipid - phosphatidylcholine. When the content of triazavirin quaternary ammonium salts in the film is less than 20 wt.%, The difference in lipid content is covered by the addition of cholesterol.

Liposomes are obtained by sequentially hydrating the lipid film in a 0.9% sodium chloride solution and then extruding through an Anopore membrane with a pore diameter of 200 nm, which is carried out at a temperature of 45-50 ° C, corresponding to the melting point of the lipid film. The total solids content in the finished liposome composition is 12.5 g / l, while the hydration of the film is carried out immediately after drying, since storage of the films for 2-3 days can lead to the formation of crystalline defects.

The parameters of liposomes obtained in this way were evaluated using dynamic light scattering spectroscopy on a Malvern Zetasizer Nano SL instrument. The evaluation results confirmed that the claimed liposome composition obtained by the claimed method has the characteristics necessary for medical use, in particular, the average size of liposomes is 180-190 nm, and the polydispersity index is 0.10-0.13.

The main parameters of the claimed liposomal composition are shown in Table No. 2.

Table number 2 Parameter Value Triazavirin in the finished liposome suspension, incl. as a part of liposome 2.0-1.6 g / l Total lipid concentration in 12.5 g / l

solution The degree of inclusion of the drug 80% Average particle size 180-190 nm Polydispersity index 0.10-0.13

The implementation of the claimed invention is confirmed by examples of specific performance.

Example No. 1.

Obtaining complex salts of Triazavirin.

500 mg of Triazavirin is dissolved in 10 ml of water and added dropwise to a 5% aqueous solution of a mixture of salts consisting of 300 mg of cetylpyridinium chloride and 280 mg of hexadecyl trimethylammonium chloride. After a short stirring, precipitation occurs, which is filtered off and dried at a temperature of 45-50 ° C.

Example No. 2.

Obtaining a lipid film with a content of Triazavirin salts with Quaternary ammonium salts of less than 20 wt.% (Without the addition of cholesterol).

Samples of 250 mg of the obtained triazavirin complex, 750 mg of phosphatidylcholine (Epicuron 200) and 3 mg of a preservative, which is used as methiparaben, are dissolved in 75 ml of chloroform. The resulting solution was evaporated on a rotary evaporator in a round flask with a volume of 250 ml at an average speed of 50-60 rpm, rotor rotation and a temperature of 45-50 ° C. The resulting film was quickly cooled to 0 ° C and dried in vacuum for 6 hours in a flask protected from sunlight. After that, the finished film is stored in the refrigerator at a temperature of -18 ° C.

Example No. 3.

Obtaining a lipid film with a content of Triazavirin salts with Quaternary ammonium salts of less than 20 wt.% (With the addition of cholesterol)

Weighed portions of 150 mg of the obtained triazavirin complex, 50 mg of cholesterol, 800 mg of phosphatidylcholine (Epicuron 200) and 3 mg of a preservative, which is used metiparaben, and 1 mg of a mixture of tocopherols (E306) are dissolved in 75 ml of chloroform. The resulting solution was evaporated on a rotary evaporator in a round flask with a volume of 250 ml at an average speed of 50-60 rpm, rotor rotation and a temperature of 45-50 ° C. The resulting film was quickly cooled to 0 ° C and dried in vacuum for 6 hours in a flask protected from sunlight. After that, the finished film is stored in the refrigerator at a temperature of -18 ° C.

Example No. 4

Lipid film hydration

After its preparation, the lipid film is poured into 50 ml of a 0.9% sodium chloride solution and placed on a shaker-type mixing device. Hydration is carried out for 3 hours at an average speed of 50-60 rpm. mixing device and a temperature of 45-50 ° C. The resulting suspension of multilamellar vesicles is centrifuged to separate large agglomerates at revolutions of 1500-2000 min ^-1 .

Example No. 5

The formation of liposomes.

The resulting suspension of vesicles in an amount of 20 ml is carefully pushed through an Anotop 25 ultrafiltration membrane nozzle with a pore diameter of 0.2 μm at a temperature of 40 ° C. The opalescent colloidal liposome solution obtained in this way is stored in a place protected from sunlight at a temperature of 4 ° C.

The main parameters of the liposome composition (liposome size and their polydispersity) did not change during storage for 5 months.

Claims (7)

1. Liposomal pharmaceutical composition comprising a medicinal substance, lipids in the form of phosphatidylcholine and cholesterol, quaternary ammonium ions, additives, according to the invention, the sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo is used as a medicinal substance [5, 1-c] -1,2,4-triazin-7-one, a dihydrate introduced into liposomes in the form of complex salts formed with quaternary ammonium salts, which are used cetylpyridinium chloride and hexadecyl trimethylammonium chloride, with the total content of complex salts at in an amount of more than 20 wt.%, phosphatidylcholine is used as lipid, with a content of less than 20 wt.% - phosphatidylcholine and cholesterol, while the components are in a certain ratio, wt.%:
2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one sodium salt, dihydrate - cetylpyridinium chloride 7.5-12.5 2-methylthio-6-nitro-1,2-4-triazolo [5,1-s] -1,2,4-triazin-7-one sodium salt, hexadecyl dihydrate trimethylammonium chloride 7.5-12.5 phosphatidylcholine 75.00-80.00 cholesterol up to 5.00 additives 0.05-0.4. 2. The liposomal pharmaceutical composition according to claim 1, characterized in that the phosphatidylcholine is Epicuron 200. 3. The liposomal pharmaceutical composition according to claim 1, characterized in that as additives use antioxidants and preservatives, 4. The liposomal pharmaceutical composition according to claim 3, characterized in that the antioxidants and preservatives are used in the following ratio, wt.%: Antioxidants - 0.05-0.1; preservatives - 0.1-0.3. 5. The liposomal pharmaceutical composition according to claim 1, characterized in that a mixture of tocopherols (E306) is used as antioxidants, and methylparaben, for example, is used as preservatives. 6. The method of obtaining the liposomal pharmaceutical composition according to claim 1, comprising introducing the drug into the liposome, characterized in that the drug substance, which is used as the sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [5, 1-c] -1,2,4-triazin-7-one, the dihydrate, is introduced into liposomes in the form of complex salts formed with quaternary ammonium salts, which are used cetylpyridinium chloride and hexadecyl trimethylammonium chloride, while the resulting complex salts are dissolved in non-polar solvent and mixture ivayut with the lipids and additives, after which the solvent was removed, and the resulting lipid film is cooled, dried in vacuo and hydrated to form a suspension of multilamellar vesicles, from which by ultrafiltration, carried out simultaneously with sterilization at a temperature of 45-50 ° C, forming liposomes. 7. A method for producing a liposomal pharmaceutical composition according to claim 6, characterized in that phosphatidylcholine and cholesterol are used as lipids.

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