RU2120302C1 - Immunomodulating agent - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: immunomodulating agent is a matrix no containing the specified antigens or antigenic determinants but containing at least one lipid and one saponine, preferably quil-A. Saponines from beta-Amirine type with carbohydrate fragments 8-11 (molecular mass are 1988, 2150 and 1862 Da) are used as a saponine source. EFFECT: development of new adjuvants. 6 cl, 7 ex, 5 tbl, 14 dwg

Description

 The invention relates to a matrix comprising at least one lipid and at least one saponin with immunomodulating action, a method for producing this base, a vaccine and a medical kit containing them and new saponins for input into the base (matrix), and a method for producing these new saponins.

 Currently, modern technological methods can be obtained many microbial and viral antigens. However, their full potential in the vaccine cannot be realized unless they are administered together with an effective adjuvant, a tool that does not increase the antibody response and / or the immune response associated with the cells.

 The only adjuvants currently approved for human use in most countries are alumina hydrate and aluminum phosphate, which have been used for many years to enhance antibody responses, for example, to diphtheria and tetanus toxoids. Although these adjuvants are suitable for many vaccines, studies have shown that in experimental animals it is often more effective to display antibody responses and cell-mediated immunity Freud's complete adjuvant (FCA) and Quil A. In practice, they are often required for protection. However, FCA causes the formation of granulomas at the points of injection, which makes it unsuitable for the preparation of vaccines for humans and for veterinary use. In fact, even aluminum hydroxide can lead to granuloma-like reactions at the injection points. For these reasons, many attempts have been made to develop adjuvants with FCA efficacy, but without unwanted side effects.

 In patent applications EPC NN 83850273.0 and 85850326.1, immunogenic complexes between antigenic determinants with hydrophobic sites and glycosides are described, and among them are triterpensaponins and especially Quil A, the so-called search complexes. In such a desired quantity, the amount of Quil A can be approximately 10-100 times less, and the same antigenic effect is created as in the case when Quil A in free form is mixed with antigen.

 European patent application 87200035.1 states that the presence of an antigen is not necessary for the formation of the basic artisan structure, it can be formed from sterol, such as cholesterol, phospholipid, such as phosphatidylethanolamine, and glycoside, such as Quil A.

 It was found that the phospholipid is not necessary to obtain the main artisanal structure that does not contain antigen. Instead, sterol, such as cholesterol, in combination with a glycoside, such as Quil A, are the main structural components assembled into a complex that reproduces a typical cell-like search structure, the so-called base. It was also determined that the base has immunomodulatory effects, such as an adjuvant or immunosuppressive effect.

 The present invention relates to a complex formed between at least one lipid, such as sterol, preferably cholesterol, and one or more saponins, such as triterpensaponins, especially Quil A or their subcomponents that are not a lipid vesicle, without directed antigens or antigenic determinants, for use as an immunomodulatory agent. Thus, in this case, the integration of any antigenic component is not carried out, as is the case in the desired one. This base has an adjuvant effect and can be used in admixture with one or more antigens, preferably in multidimensional form.

 In this desired base (matrix), integration of other adjuvants with hydrophobic sites is also possible. The introduction of other lipids may be necessary to accelerate the inclusion of other adjuvants. Thus, the present invention also encompasses a complex comprising lipids and adjuvants other than cholesterol and saponins. Such complexes contain a matrix consisting of cholesterol and saponin, preferably Quil A and its subcomponents, one or more other adjuvants and one or more lipids other than cholesterol. They are preferably not lipid vedicles or liposomes and have a very specific structure when studied using an electron microscope.

 Liposomes have been described in the literature, and their general structure is well known to biologists. Liposomes are Vedicles, including one or more consecutive rows of lipid layers forming an onion structure, separated from each other by an aqueous substance.

 This base can be introduced by injection into an animal or human body as a mixture with antigen in multidimensional form. As an option, the base and antigen may be injected separately. In this case, the best results are obtained if the base and antigen are introduced in those areas that drain into the same lymph gland. When the adjuvant is in the base in a multidimensional form according to this invention, the dose of the adjuvant may be reduced compared to the case when the adjuvant is injected separately in a one-dimensional form or in an undefined form. This means that toxic side effects caused by adjuvants during normal use, for example when they are injected as such, can be reduced or eliminated. However, the adjuvant dose cannot be reduced to the same extent as in the desired complexes according to the above patent applications.

 When using the adjuvant in the base according to the present invention, the antigen does not integrate in the same particle as the adjuvant, as occurs in the desired particle according to the above EPC patent applications. This means that you can use the antigen without amphiphatic properties or antigens that do not cause effects on hydrophobic sites. As an example, it can be pointed out that some viruses do not have amphiphatic proteins, for example picornavirus, adenovirus or parvovirus, but they are in the form of a submicroscopic particle with an antigen representing several copies (i), i.e. multimers. It is more practical to introduce such viruses together with a new adjuvant complex than to combine hydrophobic groups with them or create hydrophobic groups by other means (for example, partial denaturation) and integrate them into the desired particle.

 Typically, this base contains sterol, preferably cholesterol, and one or more saponins in a paint ratio of about 1 and 1, or in a weight ratio of about 1 to 5. These complexes have an open spherical structure consisting of round elements or fragments of a spherical structure observed by electron a microscope. Their sedimentation coefficient is approximately 20S.

 When integrating other adjuvants, the lipid-adjuvant base usually contains sterol and saponin in a molar ratio of about 1: 1, and other adjuvants and lipids together with each other comprise about 1 mol. In the case of such a base, the molar ratio of sterol-saponin: another adjuvant and lipids is about 1: 1. Thus, the molar ratio of sterol: saponin, other adjuvants and other lipids is 1: 1: 0.1-1, i.e. an additional lipid or adjuvant may be present at the base until their molar ratio (or the sum of their molar ratios) is half that of the present saponin and sterol.

 The structure determined by means of an electron microscope is the same as that of the search and base (see Fig. 1). The sedimentation coefficient, which depends on the density of the material introduced into the base, is approximately 12-22 S for bases containing cholesterol, saponin, other adjuvants and lipids.

Saponins can be any hydrophobic saponin, such as, for example, described in R. Tshesche and Welf. Chemie und Biologie der Saponin in Fortschkitte der Chemie Organisher Naturstoffe, published by W. Herz, H. Grisebach, GWKirby, Volume 30, 1973, especially highly polar saponins, mainly polar triterpensaponins, such as polar acid bisdesmosides, for example saponin extract from Kvallagabark Araloside A, Chikosetsusaponin IY, Calendula-Glycoside S. Chikosets-susaponin U, Achirantes-Saponin B, Calendula-Glycoside A, Araloside B, Araloside C, Putrangia Saponin III, Bershamidonapaponis , Trihoside B, Saponazide A, Trihoside C, Gypsoside, Nutanoside, Dianthoside C, Sap Speaking D, preferably from aestsin Aeskulus hippokastanum (T. Patt and Winkler Das therapeutisch wiersame Reinzip der Rosskastanie (Aesculus hippocastanum), Arzneimittelforchung 10 (4), 273-275 (1960),
or sapoalbin from Hyposophilus struttium (R. Vochten, P. Joos, R. Ruyssen: physicochemical properties of sapoalbin and their relation to foam stability. J. Pharm / Belg, 42, 213-226, 1968, especially saponin extract from Quillage saponaria Molina, mainly DQ, is an extract that is obtained according to K. Dalsgaakd: Saponin Adjuvants Bull off Jnt. Epiz 77 (7-8), 1289-1295 (1972) and Quil A, which is obtained according to K.Dalsgaard: Saponin adjuvants III, Archiv fur die Gesamte Virusforschung 44, 243-254, 1974. Quil A and its sub-fragments are preferred, especially fragments B2, B3 and B4B described below.

The present invention also encompasses new glycosylated triterpenoid saponins formed from Quillage Saponaria Mollin type Beta Amirin with carbohydrate groups 8-11, which have the following characteristics:
a) Substance B2 has a molecular weight of 1988. The ¹³ C NMR spectrum as shown in FIG. 5A and 6A, and ¹ H NMR as shown in FIG. 11A and 12A.

b) Substance B3 has a molecular weight of 2150 and has a ¹³ C NMR spectrum as shown in FIG. 5B and 6B and a ¹ H NMR spectrum as shown in FIG. 11B and 12B.

c) Substance B4B has a molecular weight of 1862, a ¹³ C NMR spectrum as shown in FIG. 5C and 6C, and a ¹ H NMR spectrum as shown in FIG. 11C and 12C.

 Compounds B2 and B3 have a fairly high adjuvant activity. Thus, the present invention also encompasses the use of these compounds as adjuvants. Compound B4B is used to prepare the required base. Compounds B2 and B3 having adjuvant activity may be included in this framework.

 The base can be prepared from sterol, such as cholesterol, and saponin B4B. Such a framework does not exhibit strong adjuvant activity. In order to impart an adjuvant activity to this base, it is possible or even desirable to integrate saponin B2 and / or B3 and / or any other substance with an adjuvant effect and with hydrophobic groups. If the adjuvants do not contain any hydrophobic groups, such groups can be associated with them by known chemical methods. When integrating adjuvants other than B2 or B3, they preferably comprise other lipids listed on page 13, the last paragraph and on page 14, para. 1-3.

 It is also possible to integrate immunosuppressive substances containing hydrophobic groups into the sterol-B4B base or to which such groups have been linked.

 You can also use the sterol-B4B base as an immunomodulating agent in a mixture with adjuvants, immunosuppressive substances or antigens, or mixtures thereof.

 As immunomodulating agents can be used substances that enhance, suppress or alter the immune system, such as adjuvants, suppressors, interleukins, interferons and other cytokines.

 The present invention preferably relates to a base containing sterol, especially cholesterol, B4B and either of B2 and B3, or both. When the base is made from cholesterol and Quil A, it includes B2, B3 and B4B.

 The bases can be obtained by solubilizing at least one sterol in a solvent, introducing saponin or saponins and possibly other adjuvants and lipids, after which the solvent must be removed and the base is transformed into a solution in which its components are insoluble, for example, in an aqueous solution. This can be accomplished by gel filtration, ultrafiltration, dialysis or electrophoresis. The bases should then be freed of excess sterol and Quil A by, for example, centrifugation through a density gradient, or by gel filtration. As a solvent, water, solubilizing agents or detergents described below can be used.

 The only limiting factor that occurs in the manufacture of the base is the time required under various physical and chemical conditions, the main speed limiting factor being the poor solubility of sterol, for example cholesterol, in water, in which saponins forming the base are readily soluble.

 Thus, it was shown that in the case of Quil A and cholesterol, even in the solid phase, a base formation occurs after a relatively long period of time, for example, about 1 month. Cholesterol must be brought into contact with Quil A or with its purified components. If cholesterol is converted to a colloidal aqueous suspension by ultra-sound treatment and ultra-turrax treatment, then a base forms with Quil A after about 12 hours.

 Thus, any other substance, such as a detergent, introduced into water, which increases the solubility of cholesterol in an aqueous medium, will reduce the time required for the formation of the base. Thus, it is possible to obtain a base from cholesterol, water and Quil A or its components, if cholesterol is converted to colloidal form. However, it is more practical to add a detergent or solvent.

 Saponins are preferably used in a concentration of not less than the concentration of their critical mineral formation (SMS). For Quil A, this means a concentration of not less than 0.03 wt.%.

Detergents such as non-ionic, ionic, i.e. cationic or anionic or Zwitterionic detergents, such as Zwittergene or gallic acid based detergents, which are used in excess, can be used as a stubilizing agent. Typical examples of suitable nonionic detergents are N-alkanoyl-N-alkyl-glucamines, polyhydric alcohol esters and aliphatic or araliphatic acids, and alcohols. Examples thereof are alkyl polyoxyethylene ethers of the general formula C _n H _{2n + 1} (OCH ₂ CH ₂ ) _x OH, abbreviated as C _l E _x ; alkyl phenyl polyoxyethylene ethers containing a phenyl ring between the alkyl group and the polyoxyethylene chain, briefly referred to as C _l FE _x , for example Triton X-100-tert-G ₈ E _9.6 (octyl phenol ether or polyethylene oxide), acyl polyoxyethylene esters; acylpolyoxyethylene sorbitan esters, briefly referred to as C _n sorbitan E _x , for example Tween 20, Tween 80, β-D-alkyl glucosides, for example β-D-octyl glucoside. Typical examples of suitable ionic detergents are gallic acid, for example, cholic acid, deoxycholate, cholate and CTAB (cetyltriammonium bromide). Even conjugated detergents such as taurodeoxycholate, glycodeoxycholate and glycocholate can be used. Other possible solubilizing agents are lysolectin and artificial lysophospholipids. Even mixtures of the above detergents can be used. When using the dialysis method, detergents must be dialyzed for a period not too long.

 Some surfactants significantly accelerate base formation. They include lipids of the internal biological membrane with a polar main group and a non-polar aliphatic chain, for example phosphatidylcholine (with a negative charge) and phosphatidylethanolamine (with a positive charge).

 Solubilization can also be carried out with alcohols, organic solvents or small aliphatic molecules such as heptane-1,2,3-triol, hexane-1,2,3-triol or catastrophic substances, acetic acid, such as trifluoroacetic acid, trichloroacetic acid, urea or guanidine hydrochloride.

 Ethanol, dioxane, ether, chloroform, acetone, benzene, acetic acid, carbon disulfide, MEGA-10 (N-decanoyl-N-methylglucamine) and β-octylglucoside are preferred for this use.

 Using these substances, various yields of the base can be obtained, and the overall picture is such that the more the base is formed, the higher the concentration of the detergent in the system.

 There is a technical possibility of obtaining, cleaning and sterilizing the base in any described system. Therefore, adjuvant active technical preparations may contain solubilizing agents if their chemical nature and their concentration are acceptable in the final product, for example, for vaccination purposes. However, in many cases it is necessary to remove the solubilizing agent from the base by dialysis, ultrafiltration or column chromatography. It is even possible to dilute the drug until an acceptable concentration of the solubilizing agent or detergent is reached. This preparation is diluted in water or in a physiologically acceptable solution, preferably to a concentration below SMS for the solubilizing agent or detergent in the system (preparation) used.

 The solubilizing agent can be introduced into the base in a molar ratio of sterol: saponin: additional lipid adjuvants or a solubilizing agent as 1: 1: 1, i.e. when the molar ratio of the sum of lipid, adjuvants and solubilizing agent is half that of the molar ratio of saponin and sterol.

 In addition, the solubilizing agent may remain mixed with the required base.

 For the integration of the solubilizing agent and other immunomodulatory components, there must be at least one hydrophobic zone. If they are not, then such hydrophobic zones should be paired with these components before preparing the base.

 Examples of adjuvants that can be introduced into the desired base are any adjuvants, natural or artificial, with the desired immunomodulatory effect, for example, derivatives of muramyl dipeptide (MDP), such as fatty acid, substituted MDP, threonyl analogues of MDP, amphipathic copolymers, aliphatic amines, avridine or DDA, polyanions, such as dextran sulfate, lipopolysaccharides, such as saponins (other than Quil A). ("Future Prospects for Vaccine Adjuvants," Warren HS, 1988, CRC Crif. Rev. Jmmunol., 8; 2; 83-101; Characterization of Non-Toxic Monophosphorylolipid A "1987, Johnson AG et al., Rev. Jnfect Dis. 9: 5, 5512-5516: “Developing Statement on Artificial Immunomodulators,” Berendt MJ et al., 1985, Jear Jmmunol. 193-201; “Immunopotentiating Conjugates,” Steworf-Tull DE Vaccine, 85, 3 : 1, 40-44).

 These four publications are provided here as reference bibliographic material.

 The following zwitterionic, neutral, positive and negative detergents are examples of detergents that have an immunomodulatory, especially adjuvant, effect.

 Neon block polymer surfactants, including hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP), which differ in the molar weight percent of POE and the type of binding of POP to POE (BASF Wyandotle Corp.), such as L 72, L 81 , L 92, pluronic L 101, L 121, 2531 and 31R1; octablocks T1501; B-D-octyl glucoside, cationic surfactants such as dimethyldioctadecylammonium bromide (DDA), octadecylamine (OCT) and cetyltrimethylammonium bromide (CTAB); maltosotetrapalmitate, trehalozomonomyolate, trehalose zodibenyl behenate: detergents (N-alkyl-N, N-dimethylammonio-3-propanesulfonate) Z 3-8, Z 3-10, Z 3-12, Z 3-14, Z 3-16 Calbiochem (La Jolla, CA, USA); Z 3-18, obtained by the company Serva (Heidelberg, Germany), Myry 45, Brij 52, Brij 58 (also obtained by the company Serva), and dioctyl sulfosuccinate and Tween 20, Tween 80, Triton X-100 and sodium / deoxycholate.

 These detergents can be used both as detergents and as adjuvants, and they can be introduced into the search framework.

 The following are examples of immunosuppressive agents that can be introduced into the sterol (preferably cholesterol) B4B base: cyclosporin A, diodecyldimethylammonium bromide, cationic single-chain amphiphiles with more than 10 carbon atoms, preferably more than 15 carbon atoms, double-chain amphiphiles with up to 14 carbon atoms, preferably up to 12 carbon atoms.

 In the case where the desired adjuvant or immunosuppressive agent does not possess the desired hydrophobic properties, it must be modified so as to include a hydrophobic site for insertion into the base.

 Hydrophobic groups that may be conjugated to non-hydrophobic adjuvants are straight, branched, saturated or unsaturated aliphatic carbon chains containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 carbon atoms, such as lipids, preferably containing 6.7 and 8 carbon atoms; small peptides containing 1, 2, 3, 4 or 5 amino acids, preferably 2, 3, 4 amino acids selected from Trp, Ile, Phe, Pro, Tyr, Leu, Var, especially Tyr; cholinic acid, ursodeoxycholinic acid or cholesterol derivatives.

 These hydrophobic groups must be linked to a group that can conjugate to a non-hydrophobic protein, for example, such as a carboxyl, amino, disulfide, hydroxyl, sulfhydryl and carbonyl group, for example aldehyde groups.

 As the hydrophobic groups which can be conjugated, preferably selected are carboxylic, aldehyde, amino, hydroxyl and disulfide derivatives of methane, ethane, propane, butane, hexane, heptane, octane, and peptides containing Cys, Asp, Glu, Lys, preferably octanal and Tyr, Tyr, Tyr-Cys. - Asp or Glu. Hydrophobic groups with groups that can be conjugated should be dissolved in water using, for example, solubilizing agents and detergents mentioned above, or hydrochloric acid, acetic acid, 67 vol.% Acetic acid, liquid caustic, ammonia, depending on of which substance should be dissolved. The pH in this case is brought to neutral without preliminary precipitation of the substance; however, there must be a guarantee that a pH value is not obtained that denatures the protein with which the hydrophobic group must bind.

 Hydrophobic groups with a carboxyl group as a conjugating molecule can conjugate to adjuvants via water-soluble carbodiimides or mixed anhydrides. In the first case, the carboxyl group is activated at a pH value of 5 with carbodiimide and is mixed with a protein dissolved in a buffer with a pH of 8 with a high phosphate content. In the latter case, the carboxyl compound is reacted with isobutyl chloroformate in the presence of triethylamine in dioxane or acetonitrile, and the obtained anhydride is introduced into the protein at a pH of 8 and 9. You can also convert the carboxyl group with hydrazine to hydrazide, which together with aldehydes and ketones in oxidized periodate sugar molecular units in a protein gives hydrazone bonds.

 Amino groups with nitrous acid at low temperature can be converted into diazonium salts, which give azo bonds with Tyr, His and Lys.

 Hydroxyl groups with succinic acid anhydride can be converted into semi-succinate derivatives, which can conjugate as carboxyl groups.

 Aldehyde groups can react with amino groups in the protein to Schiff base.

 Some mating groups and mating methods are described in Journal of Jmmunological Methods, 59 (1983), 129-143, 289-299, Methods in Enzymolog, Volume 93, pp. 380-33, and Analitical Biochemistry, 116, 402-407 , 1981, which are cited here as a bibliographic reference material.

 Lipids other than styrene can be fats or substances similar to fats, such as triglycerides or mixed triglycerides containing fatty acids containing up to 50 carbon atoms, for example saturated fatty acids including 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 carbon atoms, for example, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, behenic acid, lignoceric acid Or unsaturated fatty acids containing up to 30 carbon atoms such as hexadecenoic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid; hydroxy fatty acids such as 9,10-dioxistearic acid, unsaturated hydroxy fatty acids such as castor oil, branched chain fatty acids; glycerol ethers, paraffins, for example esters of higher fatty acids and monobasic alcohols; phospholipids, such as derivatives of glycerolphthalates, for example derivatives of phosphatidic acids, for example lectin, cephalin, inositol phosphatides, spingosine derivatives containing 14, 15, 16, 17, 18, 19 and 20 carbon atoms; glycolipidoisoprenoids, sulfolipids, carotenoids, steroids, sterols, cholestanol, caprostanol, phytosterols, for example stigmasterol, sitosterol, mycosterols, for example ergosterol, bile acids, for example cholic acid, deoxycholic acid, chenodeoxycholic acid, vitro, litho mixtures thereof.

 These and other useful lipids are described in: Lipid Biochemistery and introduction, Ed. M.J. Gurr, A.J. James, 1980, Charman and Hall, London, New York, University Rress Cambridge, which is incorporated herein by reference.

Holesterolfosfatidilholin preferably used, liposomes, or Intralipid ^® (Oleum Soga fraction of 200 g, a fraction Lechitinum vitello Ovi 12 g, glycerol 22.5 g, and H ₂ O to a total volume of 1 liter).

 Lipids can be introduced at any stage in this process, preferably before the introduction of saponin, but can also be entered after the introduction of saponin.

 The base is best obtained by dialysis, as described below.

Cholesterol dissolved in 20% MEGA-10 or any other suitable detergent, preferably a detergent that can be removed by dialysis, for example in β-octyl glucoside (in H ₂ O or a suitable buffer) is mixed with five times the amount of Quil A (solid or dissolved in water or a suitable buffer, for example PBS). This mixture is widely dialyzed through PBS, first overnight at room temperature (since MEGA-10 precipitates at + 4 ^o C), then at + 4 ^o C. The mixtures are purified from excess Uvil A and cholesterol by precipitation, for example, in 30% ( w / w) sucrose (e.g., TST 41.13 rotor, 18 hours, 39,000 rpm, 10 ^° C). The precipitated mixtures are dissolved in PBS (or any other suitable buffer) and the concentration is adjusted to 1 mg / ml).

 This base can be used as an immunomodulatory substance. It can be used as a potentiating agent for an immunosuppressive substance or adjuvant, or mixed with the base or intervened in the base.

 A base containing sterol, such as cholesterol, saponins, adjuvants, and other lipids if desired, can be used as an adjuvant. It can be used to potentiate the antigenic effect of any antigen or antigenic determinants of a pathogenic organism or any of their fragments or subunits or fragments or subunits formed from them. So, it can be used as an adjuvant for those antigens that integrate into the target. Such antigens are described in patent applications EPC NN 83850273.0 and 85850326.1, considered here as reference materials. So, this base can be used as adjuvants together with antigens or antigenic determinants formed from viruses with or without a shell, bacteria, protozoan organisms, mycoplasmas, mollusks, or together with such complete organisms. In addition, antigens or antigenic determinants can be hormones, enzymes, carbohydrates and carbohydrate-containing structures, such as lipopolysaccharides, peptides or proteins, or recombinants thereof.

 The present invention also encompasses medications for the treatment of humans or for veterinary use, characterized in that they contain at least one base and one or more antigenic or immunosuppressive substances, and a pharmaceutically acceptable carrier in a mixture or in separate compartments.

 The present invention also encompasses vaccines comprising a base, one or more antigens, and a pharmaceutically acceptable carrier.

 Further, the present invention encompasses a kit comprising the specified therapeutic drug or vaccine.

 A detergent used in the preparation of the base may be present in some medicinal preparations or vaccines, if the detergent is acceptable for the product.

 Further, when describing immunostimulation experiments, the effect of the new adjuvant complex of the present invention will be illustrated.

 1. Comparison of the immunogenic effects of antigens presented as isomes, minella or micelles plus a new framework.

The mice were immunized with the envelope protein from the influenza virus in the form of the desired complex, minella or minella together with the new complex according to this invention (the so-called base). The immune response was assessed by antibody determination by enzyme-linked immunosorbent assay (EL ISA) 15, 30, 44 and 50 days after injection. The following injections were carried out:
1. 5 μg of Micell + 0.1 μg of base were mixed and injected into the left front leg.

 2. 5 μg of Minell + 0.1 μg of base were injected individually into the right and left forelegs, respectively.

3. 5 mcg minell
4. 5 μg of the target were prepared according to EPC 83850273.0 (see table. 1).

 No side effects in the form of local reactions were found.

 Based on this experiment, we can conclude that the envelope protein from the influenza virus in the form of a target or micelles plus a base gives the highest antibody titers. The base may be present in very low doses, and yet it will have an adjuvant effect. In order to achieve the adjuvant effect in mice, Quil A in free form is required with a dose 100 times the dose of the base, i.e. 10 μg. At this dose, Quil A begins to produce local adverse reactions. In order for the base to have a clear adjuvant effect, the antigen in multidimensional form must be injected into the same place, for example, in the foot as the base, i.e. the injected complex of the adjuvant base and the antigen must be in the same place, in other words they must injected into the same lymph gland.

 2. Comparison of the immunogenic effects of the envelope protein from the influenza virus in the form of an iscoma or micelle with or without a base or diferitoxoid (DT).

Shell protein was injected into the body of mice in the following forms:
1.5 μg iskoma + 5 μg DT
2.5 micrograms
3.5 μg micelles
4.5 μg micelles + 0.01 μg base.

 Antibody response in envelope proteins was determined in serum by enzyme-linked immunosorbent assay (ELISA). The following results were obtained (see table. 2).

 No visible side effects in the form of local reactions were found.

 It can be concluded that the envelope protein from the influenza virus in the form of isomes or micelles plus the adjuvant complex (base) of the present invention gives the highest antibody titers. The dose of the base can be kept very low, for example 0.1 μg, and yet it will have a significant adjuvant effect.

 3. Comparison of the immunogenic effects of diphtheria toxoid DT in one-dimensional form, one-dimensional DT + iscoma containing protein coat from influenza virus, one-dimensional DT mixed with Quil A, and cholesterol and at the same time DT plus adjuvant complex (base) according to the present invention.

 In this experiment, diphtheria toxoids are used as a model antigen in one-dimensional form.

Diphtheria toxoid is injected into the body of mice in the following forms:
1.5 μg DT (diphtheria toxoid).

 2.5 micrograms of DT + 5 micrograms of target.

 3. 5 μg DT +0.5 μg Quil A + 0.1 μg CL (cholesterol).

 4.5 μg DT + 0.1 μg base (see table 3).

 The immunogenic response to DT is very low in all groups. The best result is obtained in mice immunized with diphtheria toxoid plus the base of the invention.

 From the experiments performed as described above, it can be concluded that the best results are obtained when the base of the present invention is used together with the antigen in a multidimensional form. Thus, the framework of this invention gives very good results as an adjuvant compared, for example, with Quil A in free form. For example, it should be noted that Quil A is effective as a free-form adjuvant in doses of, for example, 10 μg for mice, 50 μg for guinea pigs and 1 mtg for cattle. The practical volume for injection of the vaccine is 1 ml for small animals and 2 to 5 or 10 ml for large animals. When SMS (critical micelle concentration) for Quil A is 0.03%, 1 ml will produce 300 µg when injected with 1 milliliter. However, after the injection, due to the dilution effect, the concentration will become lower than SMS, and the micelle will become unstable.

 However, according to the present invention, saponin molecules and especially Quil A will bind together with cholesterol molecules in such a way that a relatively stable complex will be formed in very low concentrations. This complex is effective as an adjuvant in a dose that corresponds to 0.1 μg Quil A, that is, 100 times less than when Quil A is present in free form.

The figures illustrate:
FIG. 1. Image of an electron microscopic picture of a typical base.

 FIG. 2. The ultraviolet spectrum lines for Quil A. subfractions

 FIG. 3. Separation of Quil A and its sub-fractions by high-resolution thin-layer chromatography (HPTLC).

 FIG. 4. FAB mass spectra of new substances that meet this invention.

FIG. 5 and 6. Spectra ¹³ C NMR (2 zones) of new substances.

FIG. 7, 8 and 9. Full ¹³ C-NMR spectra, respectively B2, B3 and B4B.

 FIG. 10. β-amyrin skeleton.

FIG. 11 and 12. ¹ H NMR spectra of new substances.

FIG. 13. Parts of the spectrum shown in FIG. 7 and 8; and
FIG. 14. Two-dimensional NMR spectrum for substance B3.

 The present invention will now be described with reference to the following example.

 Example 1

 Basis (Cholesterol-Quil A complex).

1 mg of cholesterol dissolved in 20% MEGA-10 (in H ₂ O) was mixed with 5 mg of solid Quil A. Quil A was dissolved and the mixture was widely dialyzed through PBS, first overnight at room temperature, then at + 4 ^o C The search bases were purified from excess Quil A and cholesterol by precipitation in 30% (w / w) sucrose (TST rotor 41.13, 18 hours, 39000 rpm, 10 ^° C.). The precipitated bases were dissolved in PBS, and the concentration was adjusted to a value of 1 mg / ml (as measured by a small amount of ³ H-cholesterol).

 Example 2

 MDP (muramyl dipeptide, Sigma, adjuvant peptide) was conjugated to phosphatidylethanolamine (PEA) using N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride as described by Lefrancier et al., 1977 (Lefrancier P.rien, Choay J., Deray J., Deray J., Deray M., Lederman I. 1977, Int. J. peptide Protein Res, 9, 249-257).

In 1 mg of cholesterol (in 20% MEGA-10 in H ₂ O), an equal amount of MDP-PEA (in MEGA-10 or dimethyl sulfoxide or some other water-miscible solvent) was injected, an equal amount of phosphatidylcholine and 7 mg of Quil A (small excess compared to 5 mg, which are required for the formation of 1M). After a short incubation period at room temperature (15-30 minutes), the mixture was widely dialyzed using PBS (room temperature 4-12 hours, then + 4 ^o C).

 After cessation of dialysis, the basal complexes with the integrated additional adjuvant were purified from excess Quil A by precipitation in 10% sucrose.

 Example 3

In 1 mg of cholesterol (in 20% MEGA-10 in H ₂ O), an equal molar amount of Avridine (N, N-dioctadecyl-N ', N'-bis (2-hydroxy-ethyl) propanediamine) (in MEGA-10 or dimethyl sulfoxide) was administered or any other solvent miscible with water), an equipolar amount of phosphatidylcholine and 7 mg Quil A (a slight excess compared to 5 mg, which are required for the formation of 1M). After a short incubation period at room temperature (15-30 minutes), the mixture was widely dialyzed using PBS (room temperature 4-12 hours, then + 4 ^o C).

 After cessation of dialysis, the basal complexes with the integrated adjuvant administered were purified from Quil A and the adjuvant by precipitation in 10% sucrose (the same method as described on page 14, last paragraph).

 Example 4

In 1 mg of cholesterol (in 20% MEGA-10 in H ₂ O), an equal amount of DDA (dimethyldioctadecylammonium bromide) was introduced (in MEGA-10 or dimethyl sulfoxyl or in some other water-miscible solvent), an equipolar amount of phosphatidylcholine and 7 mg Quil A (a slight excess compared to 6 mg, which are required to form the base). After a short incubation period at room temperature (15-30 minutes), the mixture was widely dialyzed using PBS (room temperature 4-12 hours, then + 4 ^o C).

 After cessation of dialysis, the base complexes with an additionally introduced integrated adjuvant were purified from excess Quil A and the adjuvant by precipitation in 10% sucrose (the same method as described on page 14, last paragraph).

 Example 5

 2 grams of MEGA was introduced into 10 ml of water until 200 mg of cholesterol was administered, and the cholesterol was dispersed by exposure to ultrasound / ultraturrax. This mixture in an amount up to 1.6 ml could be introduced into 10 ml of a 2% solution of Quil A. The reaction mixture was completely clarified after less than 1 hour, and this indicated that all cholesterol had reacted. Using an electron microscope, it was possible to observe that the concentration of the substrate is very high, even if the concentration of the detergent in this case is 10%. Removing the detergent by dialysis or ultrafiltration did not quantify the number of particles in the base, and the base solution remained completely clear.

 This experiment showed that the formation of the base occurs in the presence of surfactants in the reaction mixtures, and that the complete formation of the base is carried out at very high concentrations of detergent.

 Example 6

 Obtaining B2, B3 and B4B (components of Quil A) according to this invention.

 5 grams of Cortex quillajae (Nordiske Droge of KemiKalieforretning, Copenhagen, lot N 8372) and 50 ml of distilled water were stirred using a magnetic stirrer for 3 hours at room temperature. The liquid phase was separated by a Buchner funnel using filter paper, and purified by filtration through a 0.22 μm Metricel Gelman membrane. This extract contained 2.5% dry material.

 This crude extract was dialyzed using 200 volumes of distilled water in a 20/32 seamless Wisking tube for 48 hours, with a water change after 24 hours. This extract was gazovan Da.

 Obtained, as indicated above, the dialyzed extract was subjected to ion exchange chromatography. A column was prepared with DEAE cellulose balanced with 0.1 mol. Tris-HCl, pH 7.5 (Whatman DE52) in column K 9/15 (Pharmacia Fine Chemicals). The material of this layer was balanced with a 0.1 Mole Tris-HCl buffer, pH 7.5. The column was eluted either in stages or with a linear salt gradient at a flow rate of 60 ml / h using a peristaltic pump. 50 ml of DQ was injected into this column and 300 downward fractions were collected (equivalent to about 5 ml). Under these conditions, some of the substances in the DQ passed unbound through the column, as will be seen in FIG. 2A (peak A). Elution was carried out until no absorption of ultraviolet rays was detected. The spectral absorbance of the effluent was measured at 280 nm by the Uvicord II system (LKB-Produkter), and fractions were collected by Golden Retriever (ISCO). At this point, a buffer containing 0.2 mol of NaCl was added, which constitutes the initial buffer. As can be seen in FIG. 2A, peak B elutes. However, some substances are still bound to the layer material to such an extent that elution is difficult even with concentrated NaCl. These are substances that impart a brownish color to DQ, while peaks A and B are only slightly colored or correspondingly completely colorless. In the next purification step, peak B was collected and subjected to exclusion gel chromatography on thin Sephadex G50, equilibrated with phosphate buffer M / 50, pH 7.5 in column K 16/70, eluting with a flow rate of 10 ml / hour. Desalination was carried out in Sephadex G25 medium in column K 16/40. Elution was carried out using a hydrostatic head of 50 cm. As can be seen in FIG. 2B, the ultraviolet lines have 3 peaks. Peak C was eluted into the void volume (as determined by Dextran Blue 2000, Pharmacia Fine Chemicals), and peak E was eluted into the total volume of the column (also determined by potassium chromate). Peak D is well separated from peaks C and E, but, as can be seen in FIG. 2B, the presence of a shoulder indicates that peak D consists of at least two substances.

 Then peak D was collected and subjected to a new separation on DEAE cellulose. The initial conditions are the same as in the first experiment with ion exchange, and as a result, all the material is adsorbed in the column. Further, the elution was carried out with a linear NaCl gradient increasing from 0 to 1 mol. (making up the initial buffer) in a volume of 300 ml. The result of this experiment is shown in FIG. 2C. Peaks F and G were found in the ultraviolet spectrum lines. F is clearly separated and is the only one substance (Section 3.3), but peak G could not be isolated because it is contaminated with F. In order to assess the homogeneity of peak F, it was collected, desalinated on Sephadex G25 and re-chromatographed in an identical experiment. As seen in FIG. 12, a single symmetrical peak (H) was detected at peak F.

 Any of these fractions (as well as DQ extract) can be further purified as follows.

Fraction H was lyophilized and dissolved in chloroform / methanol / H ₂ O (60: 40: 9, v / v / v). Then 200 mg of this solution was introduced into a high pressure liquid chromatography (HPLC) column (4.5 x 50 cm) filled with silicic acid. Iatrospheres RS-8060 (Jatron Labs, Tokyo, Japan). We used a pump operating at a speed of 5 ml / min for pumping a total of 2 L of solvent, collecting 200 fractions of 10 ml with a gradient of chloroform / methanol / H ₂ O (from 60: 40: 9 to 50:40:10). Fractions were analyzed by thin layer chromatography as follows. 2 μl of each second fraction was analyzed by developing plates of thin layer liquid chromatography (TLC plates) (HPTLC, Merek, Bodman Chemicals, Gibbstown NJ) in chloroform / methanol / 0.2% CaCl ₂ (50:40:10 v / v / v), and glycosides were determined by green staining with an anisaldehyde reagent (acetic acid / sulforic acid / paraanisaldehyde (98: 2: 1). The initial product Quil A was used as the standard for the value of R _f (see Fig. 3, which shows the separation by thin layer high pressure liquid chromatography: line 1, Quil A (fraction H); whether Adjustment 2, B1; line 3, B2; lane 4, B3; lane 5, B4A; and lane 6, B4B).

Fractions accompanying B2, B3 and B4B, having identical R _f values, are collected and analyzed for purity by thin layer chromatography. These crude fractions usually must be chromatographed twice in order to be sufficiently pure. Fractions B1 and B4A are inactive and therefore are not further separated.

The components thus enriched are further purified in a high pressure liquid chromatography (HPLC) column (21.2 x 250 mm) filled with spherical silica particles (Zorbax Si, Dupont, Wilmington, DE). 40 mg of the enriched fraction B2, B3 or B4B dissolved in 1 ml of chloroform / methanol / H ₂ O (60: 40: 9, v / v / v) are introduced into the column. A pump is used, operating at a speed of 3 ml / min to pump a total of 0.9 l of solvent, 300 fractions of 3 ml are collected using a gradient of chloroform / methanol / H ₂ O (60: 40: 9 - 50:40:10 r / v /about). Fractions are analyzed using glass-backed HPLTC plates, such as those indicated above. The purified fractions are collected and evaporated to dryness in a rotary evaporator <30 ^o C, dried in a desiccator and kept at <-20 ^o C.

 About 20-25 cycles of this purification step were carried out, that is, using (20-25) x 200 mg = 4-5 g of Quil A starting material, including repeated chromatography to prepare 1 gram of fraction B3. The output of B2 and B4B was approximately 40% of the output of B3.

 Thus prepared components B2, B3 and B4B were analyzed as follows.

 a) Mass spectrometry.

 Negative FAB mass spectrometry was performed, FIG. 4 and positive FAB mass spectrometry (data not shown) to determine the molecular weights of the purified Quil A components (B2, B3, B4A and B4B). The data presented in FIG. 1 are preliminary, and must be obtained again in a pH neutral basis, such as glycerin, and not in triethanolamine, which was used for the spectra shown in FIG. 4. This is necessary because excessive alkaline lability of these compounds has been demonstrated, pH> 8.5. The peaks at m / Z 595, 744 and 893 from the triethanolamine base should be ignored. Our B4A fraction, which contained neither an adjuvant nor an ISCOM particle, was identical to the fraction described by Komoki et al. (Structure shown in Fig. 10). Peaks corresponding to the molecular weights of the three most interesting glycosides were: m / Z 1988, B2; m / Z 2150, B3; and m / Z 1862, B4B.

b) Spectrum ¹³ C-NMR.

FiG. 5 and 6 show two regions, aliphatic carbon (8 - 45 ppm) and anomeric carbon (90 - 115 ppm), respectively, of ¹³ C-NMR spectrum for the full volume of fractions: A, B2 (20 mg); B, B3 (80 mg); and C, B4B (40 mg). All spectra were obtained in the solvent system chloroform / methanol / water (30: 60: 8, v / v / v). The triterpenoid region is well manifested (8 - 45 ppm, Fig. 6) and it is partially determined, as can be seen from the table. 4.

Partial distribution of ¹³ C-NMR signal (ppm) for the five-ring segment of β-amyrin of fractions B2, B3 and B4B (see Fig. 5) obtained in a mixture of chloroform / methanol / water (30: 60: 8, v / v / about).

 These signal distributions were carried out by emitting a large number of comparative spectra obtained in various solvents and as a result of analysis of signals independent of the solvent obtained by statistical comparison (data not shown). In FIG. Figure 6 shows the region between 80 - 148 ppm in the spectra of three compounds, A, B2; B, B3; and C, B4B, characterized by double-bonded carbon signals at 122 and 143 ppm, corresponding to C-12 and C-13, in the β-amine skeleton (Fig. 10). An anomeric carbon zone between 90 - 115 ppm shows the presence of about 9 - 10 signals corresponding to the same amount of sugar radicals in these compounds.

 Conclusion: structural differences between the fractions can be determined: A, B2; B, B3; and C, B4B, both in the spectral regions corresponding mainly to the triterpenodyne region and in the oligosaccharide parts of the molecules, respectively. More precisely, the amount of sugars cannot be determined at this point.

c) Spectrum ¹ H-NMR.

 FIG. 11 illustrates the complete proton spectrum (0-10 ppm), and FIG. 12 illustrates a partial proton spectrum (anomeric zone, 4.0 - 6.0 ppm), respectively, of fractions: A, B2; B, B3; and C, B4B. These spectra were obtained from samples (≈10 mg, ≈600 scans) dissolved in dimethyl sulfoxide (DMSO).

d ₆ / D ₂ O (98: 2, v / v). In the far left part of the spectrum (Fig. 11), at 9.4 ppm, a signal was detected from an aldehyde proton on carbon-24 (see Fig. 5). The doublet nature of this peak, the peak that was supposed to be a singlet, since it does not have adjacent protons for conjugate binding, allows us to explain the unusually complex anomeric zone, which is weakly manifested (as can be seen in the explanation to Fig. 12). This doublet may be due to the aldehyde proton in two different compounds or the chemical interaction between two different populations of the same molecule (this process is rather slow observed on the NMR time scale), which explains, therefore, the different integrals of these peaks in this doublet at different temperatures, as shown in FIG. 13 and tab. 5.

 FIG. 12 and tab. 5 (presented above) demonstrate that the relative integral of these peaks varies with temperature and that both peaks move closer to each other at a higher temperature, and they both demonstrate that there cannot be two different molecules between them, but there can be two different populations of the same molecule. This serves as an explanation of the complex anomeric zone, which is confirmed by the fact that many anomeric protons in the molecule will have double resonance due to different chemical media in these two populations. However, a series of data shows that differences in the glycosylation processes of compounds can provide a partial explanation of their structural differences (Fig. 12), showing a different number of anomeric proton signals in the spectrum. FAB mass spectrometry data for fractions B2, B3 and B4B also do not exclude the formal possibility that there are molecules of two identical sizes, very close physicochemical properties, which have the same number of sugar groups, and differ in the positions and / or sequences of bond arrangement.

Conclusion: usually one-dimensional ¹ H-NMR spectra from 8 to 10 sugar molecules containing previously unknown molecules are insufficient to determine the distribution of protons and a detailed description of the structure. To resolve all signals and determine their correct distribution over these compounds, it is necessary to use the method of two-dimensional NMR spectrum, as well as chemical decomposition of compounds for analysis. Both homonuclear ( ¹ H - ¹ H) and heteronuclear ( ¹ H - ¹³ H) spectra are COSY, TOCSY, and NOESY. A two-dimensional proton phase sensitive correlation double quantutum filtered NMR spectrum (DQFPSCOSY) for fraction B3 is shown in FIG. fourteen.

 d) General findings on structural data.

 Based on the data obtained to date, it can be concluded that active fractions that have adjuvant activity and the desired particle in Quil A contain unique glycosylated triterpenoid saponins, which differ from each other both in their triterpenoid and glycan parts. They have a structure similar to that shown in Fig. 10 and consist of a five-ring steroid skeleton of the β-amirin type and contain 8 to 11 sugar groups.

 Example 7

0.1 mg of cholesterol was mixed with ³ H-cholesterol (10 mg / ml) by dissolution in 20% MEGA-10 in H ₂ O), and 0.5 mg of B2 or B3 or B4B or mixtures thereof. This volume was adjusted to 0.5 ml, and the mixture was dialyzed using PBS on a preparation treated with ammonium molybdate (negative staining method). The dialyzed preparations were analyzed for the presence of the complex with the target structure by electron microscopy (EM) and analytical gradient centrifugation. In the study using an electron microscope, the search structure was distinguished by cateiform particles with a diameter of 40 nm, consisting of subelements with a ring structure with a diameter of 12 nm. To carry out sedimentation studies, the sample was placed in a sucrose gradient (10-50%) and centrifuged for 18 hours, at +10 ^o C, in a TST rotor of 41.14, 40,000 rpm. This gradient was collected in 18 fractions (fraction 1 = lower, and fraction 18 = upper). By localizing the activity of ³ H-cholesterol in the gradient, one can find the sedimentation constant and establish whether complexes are formed.

 B4B forms typical ischemic structures with cholesterol, but does not have strong adjuvant activity.

 B2 does not form the desired structure with cholesterol, but binds to cholesterol. B2 together with B4B forms the required structures with cholesterol. B2 has weak adjuvant activity.

 B3 binds to cholesterol but does not form the required structures, B3, like B2, together with B4B can form the desired structures with cholesterol. B3 has adjuvant activity.

Claims (6)

 1. An immunomodulating agent based on saponin, characterized in that it is the desired matrix that does not contain intentional antigens or antigenic determinants, contains at least one lipid and one saponin, preferably quil A, or one or more of its components.  2. The immunomodulatory agent according to claim 1, characterized in that it contains at least one lipid, at least one saponin and at least one adjuvant.  3. The immunomodulating agent according to claim 2, characterized in that it contains a sterol, preferably cholesterol, one or more saponins, one or more adjuvants and one or more lipids.  4. Immunomodulating agent according to paragraphs. 1 to 3, characterized in that it contains one or more immunomodulatory compounds.  5. Immunomodulating agent according to any one of paragraphs. 1 to 4, characterized in that it has an open spherical structure consisting of circular subelements or parts of a spherical structure when observed under an electron microscope and a sedimentation constant of about 12-22S. 6. Immunomodulating agent according to paragraphs. 1 to 5, characterized in that the saponins come from Quillaja Saponaria Molina beta Amirin type with 8 to 11 fragments of carbohydrate having the following characteristics: substance B ₂ has a molecular weight of 1988, ¹³ C NMR spectrum, as shown in Fig. 5A and 6A, and the proton NMR spectrum, as shown in Fig. 11A and 12A, substance B ₃ has a molecular weight of 2150 and a ¹³ C NMR spectrum, as shown in Fig. 5B and 6B, and the proton NMR spectrum, as shown in Fig. 11B and 12B, substance B ₄ B has a molecular weight of 1862, a ¹³ C NMR spectrum, as shown in Fig. 5C and 6C and the proton NMR spectrum, as shown in Fig. 11C and 11C. Priorities for items:
09/30/08 according to paragraphs fifteen;
03.22.89 under item 6.

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