RU2319506C2 - Method for preparing carrier of antigens based on lipids from sea macrophytes and triterpene glycoside cucumarioside - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to carriers of antigens. Proposed carrier represents lipid-saponin complex consisting of a mixture of triterpene glycoside, cholesterol and glyceroglycolipid. Cucumarioside A₂-2 is used as triterpene glycosides, and monogalacosyldiacylglycerides of sea macrophites are used as glyceroglycolipids. Method involves mixing solutions of cholesterol and glyceroglycolipid in chloroform, evaporation of mixture until dry under vacuum, addition of 3 weight parts of 0.4% aqueous solution of cucumarioside. Then the mixture is solubilized and the total concentration of cholesterol and glyceroglycolipid is brought about to 2 mg in 1 ml of suspension with phosphate-saline buffer at pH 7.2 followed by sonication of the prepared suspension by ultrasonic oscillation device at frequency 20 kHz for 5 min. Method provides preparing the effective adjuvant form of the carrier.

EFFECT: improved preparing method of carrier.

6 cl, 8 dwg, 13 ex

Description

The invention relates to biotechnology and medicine, namely immunology; a method for obtaining an effective adjuvant form of a carrier uniting a delivery system of antigens and natural mediators based on lipids from marine aquatic organisms is proposed.

The creation of a new generation of vaccines involves the use of isolated antigens that can provide a highly specific immune response to bacterial, viral pathogens and tumor cells.

Adjuvants of a lipid nature were used long before the use of mineral sorbents as carriers in vaccine preparations. Antigens immobilized on lipid carriers have high immunological activity, and the carriers themselves are biodegradable and lack of their own immunogenicity.

It is known to obtain lipid carriers in the form of emulsions. In particular, the use of plant galactolipid digalactosidiacylglycerol (DGDG) was proposed as the main component [US Pat. RU 2127124, publ. 03/10/1999,], forming in water-in-oil solutions reverse micelles and two-layer vesicles. However, oil adjuvants often with subcutaneous and intramuscular injection cause local inflammatory-necrotic reactions, and the increased viscosity makes their administration difficult. The specified emulsion does not give a significant increase in the immunogenicity of protein and other antigens, since DGDG itself does not have immunoadjuvant properties. Other disadvantages of emulsified antigens that limit their use include non-standard quality and instability during storage of the vaccine in the finished form as a result of phase separation.

The use of liposomes as vesicles for presenting antigens is known [Allison AG, Gregoriadis G. Liposomes as immunological adjuvants. Nature. 1974. N.252. P.252]. The main structure-forming component of the proposed liposomes is lamellar phosphatidylcholine. Liposome vesicles can include both hydrophobic antigenic material in the lipid bilayer and hydrophilic in the internal phase. The ability of liposomes with a built-in antigen to stimulate antibody formation, activate phagocytes and be absorbed by macrophages in various organs and tissues of the body, and ensure the delivery of drugs (antigens) to the cell is shown. Antigens in liposomal preparations can be considered as immunoadjuvants. Unlike other adjuvants, the formation of granulomas does not occur at the injection site of liposomal preparations, and even the prevention of toxic manifestations of encapsulated drugs is observed. The disadvantages leading to the limitation of the widespread use of vaccine preparations based on the proposed liposomes are:

- chemical and physical instability of vesicles;

- a relatively moderate immune response;

- inability to stimulate a cellular immune response, providing antiviral and antitumor protection.

The closest to the claimed technical solution according to the technical essence and the achieved result is an immunostimulating complex (ISCOM), which is a lipid-saponin particles, which consist of a mixture of Quil A plant triterpene glycosides, cholesterol and phospholipids [US Pat. RU 2120302, publ. October 20, 1998] (prototype). Such complexes are not lipid vesicles or liposomes and have a very specific structure, confirmed by electron microscopy. SUITS are colloidal particles consisting of regular subunits with an open spherical structure that look like rings under a microscope. The presence of antigen for the formation of the main artisanal structure is optional. An important role in the stabilization of ISCOM is played by the structural and mechanical factor caused by the formation of a strong glycoside-cholesterol complex, which, on the one hand, limits the conformational mobility of the fatty acid chains of phospholipids, and, on the other hand, obviously contributes to the development of an immune response. The embedded Quil A saponins in the carrier are powerful immunostimulants and the main formative components. The effectiveness of ISCOM allows one to reduce the dose of administered antigens by an order of magnitude, induce significant antiviral immunity and increase the level of the humoral immune response in adjuvant doses much lower than in conventional vaccines.

The main drawback of ISCOM as an antigen delivery system is toxicity after intravenous administration of the drug, the main reason for which is the relatively high hemolytic activity of Quil A saponins. Intramuscular administration of these saponins and ISCOM is associated with inflammatory and pain reactions in humans. Currently, the use of Quil A saponins as a non-specific immunostimulant in humans is limited to oral administration. Attempts to isolate from the Quil A fraction of more purified saponins, characterized by low cytotoxic properties, lead to the fact that the obtained saponins either lose their ability to form isosomal structures or do not have immunostimulating activity.

The objective of the proposed technical solution is a method of obtaining a carrier of antigens that provides increased safety (lack of toxicity, inflammatory and pain reactions) and the effectiveness of vaccines.

The problem is solved by a method of obtaining a carrier based on lipids from marine aquatic organisms, in which the structure-forming base of the lipid-saponin carrier is a non-toxic complex of tucerpene glycoside of the cucumaria cucumaria japonica with cholesterol and glyceroglycolipid from marine macrophytes.

The problem is solved in an optimal way by the method of obtaining a carrier in the form of a lipid-saponin complex, in which cucumarioside A ₂ -2 from cucumaria Cucumaria japonica (KD preparation) is used as a triterpene glycoside, the weight ratio of which to cholesterol is constant and equal to 3: 2, and monogalactosyl diacylglyceride (MGDG) from marine macrophytes, the content of which in the complex can vary in the range from 1 to 10 (in a weight ratio), is used as a glyceroglycolipid from marine macrophytes. The method involves mixing CD, cholesterol and MGDG taken in a weight ratio of 3: 2: (1-10) in the following order: mix solutions of cholesterol and MGDG in chloroform at a weight ratio of cholesterol and MGDG 2: (1-10), evaporated in vacuo until chloroform is removed and 3 parts by weight of a 0.4% aqueous solution of cucumarioside are added, the mixture is solubilized, and then the total concentration of lipids (cholesterol and MGDG) is adjusted to 2 mg in 1 ml of suspension with phosphate-buffered saline at pH 7.2. The resulting suspension is voiced by an ultrasonic disintegrator for 5 minutes at a frequency of 20 kHz.

The inventive method for the first time received an antigen carrier with high immunological characteristics, which provides increased safety of vaccines and has a structure different from the structure of known immunostimulating complexes. The obtained lipid-saponin complexes have an ultramicroscopic tubular structure with a tube diameter of about 40 nm, which is confirmed by electron microscopy (Fig. 2, pictures A, B, C, D and FIG. 3, picture A). Figure 2 shows electronic micrographs of micelles of antigen carriers obtained by changing the quantitative content of MHD in the CD system: cholesterol: MHD; A - (3: 2: 1); B - (3: 2: 4); B - (3: 2: 6); G - (3: 2: 10).

It was found that complexes with such a structure are formed only when using all three components involved in the formation of the carrier obtained by the claimed method, taken at their optimal ratio, namely, the weight content of CD: cholesterol: MGDG is 3: 2: (1-10) .

Preparation KD prepared from natural raw materials available, broth sea cucumber Cucumaria japonica, which is the sum of triterpene glycosides, the major component of which is kukumariozid A ₂ -2 (Avilov SA, LY Tishchenko, V. Structure Stonik kukumariozida A ₂ -2 - triterpene glycoside from holothurium Cucumaria japonica / Chemistry of natural compounds. 1984. No. 6. P.799-800). Its structural formula is shown in figure 1 (A).

Cucumarioside does not exhibit embryotoxic and mutagenic properties and increases nonspecific resistance to bacterial and viral infections in extremely low doses that are not toxic to the body. The observed effect of cucumarioside is associated with the stimulation of the phagocytic activity of tissue macrophages that provide nonspecific protection of the body and mediate the recognition of antigen by the immune system, affect the level of proliferative activity of stem cells, and enhance the cooperative interaction of immunocompetent cells (T and B lymphocytes) in the development of a specific immune response and probably by direct action at the stage of virus-cell interaction.

Thus, the presence of cucumarioside in the complex obtained by the claimed method allows to increase the effectiveness of antiviral vaccines - to increase their immunostimulating activity against a number of viruses and to exclude hemotoxicity.

When creating antigen carriers in order to enhance the immunogenicity of immunocompetent cells, the choice of lipid is important. The biological activity of marine glyceroglycolipids selected for the production of antigen carriers by the claimed method is reasonably associated with both the structure of the carbohydrate component and the presence of a large amount of polyunsaturated fatty acids (PUFAs). It is known that glycoglycerolipids from seaweeds (macrophytes) belonging to different phylogenetic groups differ significantly in fatty acid composition and degree of unsaturation. The composition of fatty acids of marine glycolipids is very diverse and specific to each type of algae. Based on the structure-forming properties of glyceroglycolipids, MGDG was chosen for the formation of ISKOM, the structural formula of which is shown in Fig. 1 (B). MHD used as a lipid component from Ulva fenestrata (green algae department - Chlorophyta), Ahnfeltia tobuchiensis (red algae department - Rhodophyta), Laminaria japonica (brown algae department - Phaeophyta), Zostera marina (green sea grass - Magnoliophy department) method Sanina N., Goncharova S., Kostetsky E. Fatty acid composition of individual polar lipid classes from marine macrophytes // Phytochemistry. 2004. V.65. No. 6. P.721-730. This lipid is the most unsaturated glycoglycerolipid and, depending on the source of isolation, has a wide spectrum of fatty acid composition. So, U.fenestrata and Z.marina contain up to 93%, while A. tobuchiensis and L. japonica contain up to 73% omega-3 PUFAs. According to known data, such fatty acids have an immunomodulatory effect, affecting the activation of class 1 and 2 helper T cells, stimulation of T cell proliferation and the formation of cytokines, and also have selective cytotoxicity against tumor cells, enhancing the action of cytotoxic agents. The prior art knows the effect of unsaturated fatty acids, in particular linolenic and arachidonic, on the state of the membranes of cells of the immune system, especially macrophages.

The constant weight ratio of CD and cholesterol upon receipt of an antigen carrier by the claimed method, equal to 3: 2, is determined, on the one hand, by a fact known from the prior art. With this ratio, the complex does not have hemolytic activity and does not have a toxic effect when administered intraperitoneally to experimental animals. On the other hand, the optimality of such a weight ratio of CD and cholesterol is confirmed by experimental data (examples 5, 6). Figure 3 presents electronic micrographs of micelles of antigen carriers obtained at a weight ratio of CD: cholesterol: MGDG equal to: A - (4: 2: 3); B - (4: 1: 6). The increase in CD content in the system upon receipt of an antigen carrier is irrational, because this does not lead to a change in its tubular structure (example 5, figure 3, picture A). The data obtained in example 6 (figure 3, image B) show that a decrease in the amount of cholesterol during the implementation of the method leads to the disappearance of tubular structures.

It was also found that the amount of MGDG isolated from various marine macrophytes upon receipt of an antigen carrier by the claimed method does not affect its immunological properties and morphological characteristics if its weight ratio to CD and cholesterol varies in the range (1-10), i.e. the optimal ratio of CD, cholesterol and MGDG is 3: 2: (1-10). Examples 1-4, 8-10 (Fig. 2, pictures A-D) confirm that the introduction of MHD into a mixture of CD and cholesterol in the indicated proportions promotes the formation of a lipid-saponin complex with the necessary adjuvant properties of a natural carrier and having a standard tubular structure. The increase in the content of MGDG is irrational, because however, there is no significant improvement in the immunological and morphological characteristics of the resulting antigen carriers.

The reduction in the weight content of MHD in the initial mixture to its complete absence upon receipt of the antigen carrier by the claimed method leads to the disappearance of structures visible under an electron microscope (Example 7).

The method involves mixing CD, cholesterol and MGDG taken in a weight ratio of 3: 2: (1-10) in the following order: mix solutions of cholesterol and MGDG in chloroform at a weight ratio of cholesterol and MGDG 2: (1-10), evaporated in vacuo until chloroform is removed and 3 parts by weight of a 0.4% aqueous solution of cucumarioside are added to the mixture, after which the total concentration of lipids (cholesterol and MGDG) is adjusted to 2 mg in 1 ml of suspension with phosphate-buffered saline at pH 7.2. The indicated concentration of lipids is optimal for immunological and electron microscopic studies. The resulting suspension is voiced by the ultrasonic disintegrator SONOPULS Ultraschall-Homogenizatoren HD 2070 (Germany) at a frequency of 20 kHz, power 7 W in mode 7 (0.7 sec. Operation, 0.3 sec. Break) for 5 minutes.

The properties of the antigen carrier obtained by the claimed method do not follow with obviousness from the prior art, which is confirmed by data and experimental results known from the literature.

The resulting preparations are an aqueous suspension of lipid-saponin complexes having an ultramicroscopic tubular structure with a tube diameter of about 40 nm, regardless of the source of MHD generation from marine macrophytes (examples 1-4, 8-10).

The authenticity of the drug is determined by the qualitative and quantitative composition of the structural components (cucumarioside, cholesterol and MGDG), which are the initial ones when receiving the carrier of antigens; its structure is confirmed by electron microscopy (Fig.2, 3). The drug is stable at a temperature (-4 ° C) of more than three months.

The proposed complex with subcutaneous and intraperitoneal administration to mice does not cause toxic (example 11), inflammatory, pain (example 12) and hemolytic (example 13) effects.

The use of antigen carriers obtained by the claimed method allows:

- eliminate the inflammatory, pain, toxic and hemolytic effects of vaccines;

- increase the immunogenic activity of vaccines and their effectiveness against a number of viruses;

- ensure the stability of vaccines during storage.

Examples of specific embodiments of the invention

The starting components for the synthesis of lipid-saponin complexes by the claimed method, namely triterpene glycoside cucumarioside A ₂ -2 from Cucumaria japonica (CD), cholesterol and glycoglycerolipid MGDG from marine macrophytes, are taken in a predetermined optimal weight ratio. Solutions of cholesterol and MGDG in chloroform are evaporated in vacuo to remove chloroform, a 0.4% aqueous solution of cucumarioside is added, solubilized and the total concentration of lipids (cholesterol and MGDG) is adjusted to 2 mg in 1 ml of suspension with phosphate-buffered saline at pH 7.2 ; the resulting suspension is voiced by an ultrasonic disintegrator for 5 minutes at a frequency of 20 kHz. The resulting preparations of antigen carriers are an aqueous suspension comprising particles of a complex having an ultramicroscopic tubular lipid structure; the diameter of the tubes is about 40 nm.

The preparations of the lipid-saponin complex in the above examples 1-6 were obtained according to the described method using MGDG isolated from U.fenestrata.

Example 1. Preparation of a complex preparation consisting of 3 weight parts of CD, two weight parts of cholesterol and one weight part of MGDG. 5 mg MGDG is dissolved in 1 ml of chloroform; 5 mg of cholesterol is dissolved in 1 ml of chloroform, and 4 mg of CD is dissolved in 1 ml of distilled water. 27 μl of cholesterol solution and 13 μl of MGDG solution were taken with a microdoser, evaporated to dryness under vacuum and the residue was solubilized with a CD solution, the volume of which was 500 μl. 500 μl of phosphate buffer, pH 7.2, are added to the mixture. The final lipid concentration is 2 mg per 1 ml. The suspension is voiced for 5 minutes by an ultrasonic disintegrator at a frequency of 20 kHz. The resulting preparation is an aqueous suspension comprising particles of a complex having an ultramicroscopic tubular lipid structure with a diameter of about 40 nm. Figure 2 (picture A) shows an electron micrograph of the obtained lipid-saponin complex (magnification X 50,000).

Example 2. The preparation of the lipid-saponin complex was obtained according to the described procedure with a weight ratio of the starting components of 3: 2: 4. Figure 2 (snapshot B) shows an electron micrograph of a lipid-saponin complex obtained under the conditions of example 2 (magnification X 50,000).

Example 3. The preparation of the lipid-saponin complex was obtained according to the described procedure with a weight ratio of the starting components of 3: 2: 6. Figure 2 (snapshot B) shows an electron micrograph of a lipid-saponin complex obtained under the conditions of example 3 (magnification X 50,000).

Example 4. The preparation of the lipid-saponin complex was obtained according to the described procedure with a weight ratio of the starting components of 3: 2: 10. Figure 2 (snapshot D) shows an electron micrograph of the lipid-saponin complex obtained in the conditions of example 4 (magnification X 50,000).

Example 5. The preparation of the lipid-saponin complex was obtained according to the described procedure with a weight ratio of the starting components of 4: 2: 3. Figure 3 (picture A) shows an electron micrograph of a lipid-saponin complex obtained under the conditions of example 5 (magnification X 50,000).

Example 6. The preparation of the lipid-saponin complex was obtained according to the described procedure with a weight ratio of the starting components of 4: 1: 6. According to electron microscopy, there is a disappearance of tubular structures and the appearance of filamentous structures. Figure 3 (snapshot B) shows an electron micrograph of a lipid-saponin complex obtained under the conditions of Example 6 (magnification X 50,000).

Example 7. The preparation of lipid-saponin complexes was obtained according to the described method on the basis of cucumarioside and cholesterol with their optimal weight ratio of 3: 2 (in the absence of MGDG). According to electron microscopy, cucumarioside and cholesterol in the absence of MHD do not form visible structures.

Example 8. The preparations of the lipid-saponin complex were obtained according to the described procedure with the ratio of the starting components taken in example 1, using MGDG isolated from A. tobuchiensis.

Example 9. Preparations of the lipid-saponin complex were obtained according to the described procedure with the ratio of the starting components taken in example 2, using MGDG isolated from L. japonica.

Example 10. Preparations of the lipid-saponin complex were obtained according to the described procedure with the ratio of the starting components taken in example 3, using MGDG isolated from Z.marina.

The structure of the lipid-saponin complexes obtained in examples 8-10 does not differ from the structure of the complexes obtained in examples 1-4.

Obtained by the claimed method, the antigen carrier was tested on a group of outbred mice (10 animals in the control and experimental group) for its safety in the appearance of toxic, inflammatory and pain reactions and death of animals. The hemolytic effect of lipid-saponin carriers was studied in vitro using outbred mice as a test system of red blood cells.

Example 11. A single intraperitoneal administration to the white outbred mice of the lipid-saponin complexes shown in examples 1-4, 8-10, at the rate of 100 and 200 μg of CD per mouse does not cause animal death and toxic effects within three days of observation. The tested doses of CD correspond to 5 and 10 mg / kg of body weight. The optimal pharmaceutical dose of cucumarioside is between 0.004 and 40 micrograms per kg of body weight.

Example 12. A single subcutaneous injection of the lipid-saponin complexes shown in examples 1-4, 8-10, at the rate of 100 and 200 μg of CD per mouse does not cause inflammatory and pain effects in animals during three days of observation.

Example 13. The lipid-saponin complexes described in examples 1-4, 8-10, were tested for hemolytic activity in vitro. Heparinized blood obtained by decapitation of the mouse was washed by centrifugation 2 times at 3000 rpm for 15 min at room temperature in an isotonic sodium chloride solution. A 0.5% cell suspension was obtained from the erythrocyte sediment. The studied preparations were prepared in phosphate-buffered saline, pH 7.4, at the rate of 60 μg of cucumarioside per 1 ml of lipid suspension, 10 μl was sequentially distributed into the wells of the tablet by two serial dilutions and 90 μl of erythrocyte suspension was added. 10 μl of phosphate-buffered saline was added to control wells. Incubation was carried out for 1 and 2 hours in a thermostat at 37 ° C. The hemolytic activity of the studied drugs was evaluated by visual inspection of the wells. As a result of the experiment, the minimum effective dose of the substance causing 100% hemolysis (ED ₁₀₀ ) was determined, at which a clear solution of “varnish” blood was observed. The lipid-saponin complexes shown in examples 1-4, 8-10, at doses up to 60 μg of CD in the preparation per 1 ml of 0.5% cell suspension did not show a hemolytic effect in relation to white mouse red blood cells within two hours of observation.

Claims (6)

1. A method of obtaining a carrier of antigens in the form of lipid-saponin complexes, consisting of a mixture of triterpene glycoside, cholesterol and glyceroglycolipid, characterized in that pre-mixed solutions of cholesterol and glyceroglycolipid in chloroform, the mixture is evaporated to dryness under vacuum, add 3 weight parts of 0.4% aqueous solution of cucumarioside, after which the mixture is solubilized and the total concentration of cholesterol and glyceroglycolipid is adjusted to 2 mg in 1 ml of suspension with phosphate-buffered saline at pH 7.2 and the resulting sound is voiced th suspension by ultrasonic disintegrator for 5 min at a frequency of 20 kHz. 2. The method according to claim 1, characterized in that monogalactosyldiacylglyceride (MGDG) from marine macrophytes is used as a glyceroglycolipid. 3. The method according to claims 1 and 2, characterized in that the weight ratio of cholesterol to MGDG is 2: (1-10). 4. The method according to claim 1, characterized in that cucumarioside A ₂ -2 (CD) is used as the triterpene glycoside. 5. The method according to claims 1 and 4, characterized in that the weight ratio of CD to cholesterol is constant and equal to 3: 2. 6. The method according to claims 1-5, characterized in that the obtained lipid-saponin complexes have an ultramicroscopic tubular structure with a tube diameter of about 40 nm.

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