RU2214278C1 - Adjuvant for biopreparations - Google Patents

Abstract

FIELD: medicine, biotechnology, immunology. SUBSTANCE: the present innovation deals with preparations stimulating antibody-forming and could be applied in the field of medicine and veterinary science to constructing and manufacturing highly efficient vaccines. Adjuvant for biopreparations contains distilled water, conservant and nonionogenic SAS as foundation. Additionally, adjuvant contains immunomodulator as fat- and/or water-soluble vitamins, and cosolvent, as well. The adjuvant elaborated enables to create vaccines of low toxicity, increased immunogenic activity, stability of immunogenic properties at storage and low cost price of production. EFFECT: higher efficiency. 3 cl, 9 ex, 6 tbl

Description

 The invention relates to biotechnology and immunology, in particular to drugs that stimulate antibody formation, and can be used in the field of medicine and veterinary medicine for the design and manufacture of highly effective vaccines.

 Adjuvants - substances or mixtures of substances, incl. in the form of suspensions and emulsions, causing increased formation of antibodies to complex antigens (AH), such as bacteria and viruses. Adjuvants include drugs that enhance the production of specific antibodies to an antigen by stimulating the body's immune system.

 Micellar solutions of surface-active substances (surfactants) are lyophilic dispersed systems in which, along with individual molecules, colloidal particles (micelles) are present - associates of surfactant molecules with a fairly high degree of association (the number of molecules in a micelle). When such micelles are formed in a polar solvent, the hydrocarbon chains of surfactant molecules are combined into a compact hydrocarbon core, and the hydrated polar groups facing the aqueous phase form a hydrophilic shell. A special case of micelles are formations, which are closed pseudo-cellular structures from derivatives of phosphatidylcholine, lecithin or cholesterol. Such micelles are called liposomes.

 The most common adjuvants for vaccines (bacterial and viral) are water-oil emulsions in the presence of emulsifying substances or suspensions of inorganic substances (see RF patent 2058154, IPC A 61 K 39/135, RF patent 1743027, IPC A 61 K 39/135, IPC A 61 K 39/39, application WO09701133, IPC A 61K 9/14, US patent 5312620, IPC A61K31 / 765).

For example, a traditional water-oil adjuvant for biological products (RF patent 1730763 dated 10.12.95) containing PES-3 polyethylsiloxane liquid as an emulsifier in the following proportions, wt.%:
PES - 91-94
Etherified Polyglecirin - 6-9
Known adjuvant, which as a base includes 83% of light mineral oil for emulsified vaccines and antigens (TU-38101307) and as an emulsifier of 17% anhydrous lanolin. The vaccine based on this adjuvant is a water-in-oil emulsion.

Known liposomal adjuvants for biological products (US patent 485388 from 02.26.90, US patent 108509 from 1.07.98), representing a dispersion of liposomes in water. Liposomes were prepared from dimyristoylphosphatidylcholine and cholesterol in the following proportions, mol.%:
Dimyristoylphosphatidylcholine - 70-40
Cholesterol - 60-30
However, preparations based on oil emulsions and suspensions of inorganic substances, as a rule, are prone to phase separation and reactogenic. Most of these adjuvants cause local and systemic toxic effects. In addition, preparations prepared on their basis have a high viscosity, which complicates their introduction.

 Liposomal adjuvants are not stable during storage, and the production of liposomes is expensive.

 Closest to the proposed invention is US patent 6086901 from 07/11/2000 "The use of microparticles combined with submicron emulsions such as water-oil". This preparation contains an immunological adjuvant in the form of an emulsion and the target antigen enclosed in biodegradable microparticles. The immunological adjuvant contains an oil base, water, surfactants as an emulsifier - 4-5% squalene, 0.25-0.5% polyoxyethylene sorbitan monooleate and 0.5% sorbitan trioleate, an active substance with immunomodulating properties - a derivative of muramyl dipeptide .

 However, this drug has high toxicity and insufficient immunogenic activity, the inclusion of muramyl dipeptide and the conclusion of the antigen in biodegradable microparticles requires large material costs.

 The objective of the present invention is to develop an adjuvant that allows you to create vaccines with low toxicity, increased immunogenic activity, the stability of immunogenic properties during storage and low production costs.

The problem is solved in that the adjuvant for biological products containing a base, distilled water, a preservative, according to the proposed solution as a base contains nonionic surfactants in the following ratio of components (wt.%.):
Nonionic surfactants - 5-30
Preservative - 0.05-5
Distilled Water - Up to 100
Adjuvant for biological products additionally contains an immunomodulator in the form of fat and / or water soluble vitamins in an amount of from 3 to 18 wt.%.

Adjuvant for biological products contains vitamins A, E, C as fat and / or water soluble vitamins in the following ratio, wt.%:
A - 3
E - 5
C - 10
The adjuvant for biological products additionally contains a co-solvent in an amount (wt.%) Of 1-25%.

 In the prototype there are non-inogenic surfactants, however, in a different percentage, they also serve as an emulsifier.

 The essence of the invention lies in the fact that an aqueous-organic dispersion of nonionic surfactants is used as an adjuvant. This composition allows you to add to the vaccine various lipophilic biologically active substances (vitamins, immunomodulators, etc.) that can affect the immunogenic activity of the vaccine.

The preparation is a water-organic water-surfactant dispersion system, polyoxyethylated castor oil, or polysorbates, or polyoxyethyl hydroxystearate, or fatty acid sorbitol esters as a co-solvent: dimethyl sulfoxide, 2-pyrrolidone or dimethyl acetamide or a small molecule are used as surfactants. water or an aqueous solution of biologically active substances (vitamins), for example, benzyl alcohol, or parabens, or chlorethon are used as a preservative the following content of components (wt.%):
Non-inogenic surfactants - 5-30
Preservative - 0.05-5
Co-solvent - 1-25
Distilled water - Up to 100
A study of the effect of vitamins on antibody-forming cells showed their role in stimulating lymphocytes of the T-helper link and the formation of a T-dependent immune response. In addition, vitamin E, by inactivating the free radicals of cells, inhibits the expression of anti-inflammatory cytokine genes: IL-1, IL-6, IL-8, cytokine receptors, and others. When studying the effect of vitamin A on the body, its beneficial role in stimulating antibody-forming cells was noted phagocytes of the spleen and suppression of the processes of atrophy of the thymus. Vitamin C (ascorbic acid) contributes to the stimulation of general factors of body resistance by increasing non-specific mechanisms of immunity (Immunopharmacology of trace elements. Kudrin A.V., Skalny A.V. et al. 2000).

The method of preparation of the drug (dosage form)
1. The surfactant, cosolvent and preservative were mixed at a temperature of 30-40 ^o C. The solution was weighed antigen with constant stirring. To the resulting mixture was added the calculated amount of distilled water. After solubilization of the antigen, pH control was performed and the preparation was packaged in a suitable container under aseptic conditions.

2. The surfactant, cosolvent and preservative were mixed at a temperature of 30-40 ^° C. The calculated amount of distilled water was added to the resulting solution. After the components were completely mixed, the pH was monitored, a weighed of antigen was added, and the solution was mixed until the antigen was completely solubilized. The resulting preparation was packaged in a suitable container under aseptic conditions.

 3. The same as in claim 1, with the addition of lipophilic vitamins to the surfactant / cosolvent / antigen mixture.

 4. The same as in claim 2, with the addition of lipophilic vitamins to the surfactant / cosolvent mixture.

 5. As in claim 1, with the addition of distilled water of ascorbic acid and / or other water-soluble biologically active substances.

 6. The same as in claim 2, with the addition of distilled water of ascorbic acid and / or other water-soluble biologically active substances.

 The following surfactants may be used in this dosage form: polyoxyethylene castor oil; polyoxyethyl hydroxystearate; polysorbate 80; propylene glycol monopalmitostearate and organic solvents: dimethyl sulfoxide; dimethylacetamide; 2-pyrrolidone; propylene glycol; glyceroformal; transcutol; PEG 400.

 The following are examples of ratios of components in the micellar dosage form of an adjuvant. The antigen is added in a ratio of 1: 1 to 1: 100000, depending on the desired immune properties of the vaccine.

Example 1
The optimal ratios and the most preferred types of components are given. The amounts of surfactants shown in the table have an optimal concentration and stability.

Name of component - Amount, wt.%
Polysorbate 80 or Polyoxyethyl Hydroxystearate - 20-22
Dimethylacetamide or 2-pyrrolidone - 15
Preservative - 1
Distilled water - Up to 100
Example 2
Minimum component ratios are given. The amounts of surfactants and cosolvent shown in the table have a minimum concentration.

Name of component - Amount, wt.%
Polysorbate 80 or Polyoxyethyl Hydroxystearate - 5.0
Dimethylacetamide or 2-pyrrolidone - 1.0
Preservative - 0.05
Distilled water - Up to 100
Example 3
The maximum ratios of the components are given. The amounts of surfactant and cosolvent shown in the table have a maximum concentration.

Name of component - Amount, wt.%
Polysorbate 80 or polyoxyethyl hydroxystearate - 30.0
Dimethylacetamide or 2-pyrrolidone - 25.0
Preservative - 5.0
Distilled water - Up to 100
Example 4
The optimal ratios of components and lipophilic biologically active substances are given. The amounts of surfactants and vitamins shown in the table have an optimal concentration and stability.

Name of component - Amount, wt.%
Vitamin A - 3
Vitamin E - 5
Vitamin C - 10
Polysorbate 80 or Polyoxyethyl Hydroxystearate - 23
Dimethylacetamide or 2-pyrrolidone - 16
Preservative - 1
Distilled Water - Else
Example 5
The formulation of the drug without the addition of vitamin C with optimal concentrations of vitamins A and E.

Name of component - Amount, wt.%
Vitamin E - 5
Vitamin A - 3
Polysorbate 80 or polyoxyethyl hydroxystearate - 23.0
Dimethylacetamide or 2-pyrrolidone - 16.0
Cremophor - 20
Co-solvent - 15
Preservative - 1
Distilled water - Up to 100
Example 6
The formulation of the drug containing the optimal concentration of vitamin A and not containing vitamins E and C.

Name of component - Amount, wt.%
Vitamin A - 3
Polysorbate 80 - 22
Dimethylacetamide or 2-pyrrolidone - 15
Preservative - 1
Distilled water - Up to 100
Example 7
The formulation of the drug containing the optimal concentration of vitamin E and not containing vitamins A and C.

Name of component - Amount, wt.%
Vitamin E - 5
Polyoxyethylene Castor Oil - 24
Dimethylacetamide or 2-pyrrolidone - 15
Preservative - 1
Distilled water - Up to 100
Example 8
The maximum ratios of components and biologically active substances are given. The amount of surfactant co-solvent and preservatives shown in the table have the highest possible concentration.

Name of component - Amount, wt.%
Vitamin A - 4.5
Vitamin E - 7.0
Vitamin C - 10
Polyoxyethyl Hydroxystearate or Polysorbate 80 - 30
Dimethylacetamide, or 2-pyrrolidone, or transcutol - 20
Preservative - 2
Distilled water - Up to 100
Example 9
Minimum ratios of components and biologically active substances are given. The amount of surfactant co-solvent and preservatives shown in the table have the lowest possible concentration.

Name of component - Amount, wt.%
Vitamin A - 0.5
Vitamin E - 1.0
Vitamin C - 0.5
Polyoxyethyl Hydroxystearate or Polysorbate 80 - 5.0
Dimethylacetamide, or 2-pyrrolidone, or transcutol - 1.0
Preservative - 4.0
Distilled water - Up to 100
The tests showed that a decrease or increase in the components in the formulation leads to a qualitative change in the properties of the dosage form, i.e., an increase in the amount of surfactant and / or co-solvent leads to an unacceptable increase in the viscosity of the solution, a decrease in the amount of surfactant and / or co-solvent leads to liquid separation.

 The use of other surfactants and cosolvents in the above formulations did not lead to a sharp change in the physicochemical and pharmacological properties of the obtained adjuvant.

The study of the biological properties of drugs
A test of the dosage form based on the claimed adjuvant was carried out on white mice (females), rabbits (chinchilla breed), white rats. The LD50 of the preparations in Examples 1-8 was 9.0 ± 3.0 ml / kg gm.t. (intraperitoneal, mice). A solution of bovine serum albumin (BSA) and an extract from the bacterium Y. Pseudotuberculosis (AGY) were used as model antigens. Mixtures of antigens with Freund's complete adjuvant (PAF) were used as a positive control. (Freund's complete adjuvant is one of the strongest adjuvants used in immunology. However, due to frequent complications, this adjuvant is used only in laboratory practice.) Antigen solutions in physiological saline (FS) were used as a negative control.

Experiment 1
The study was carried out on sexually mature female rats in an amount of 60 pieces. The objects were divided into ten groups of 6 animals in each group and were immunized as described in table 1. BSA was taken as a model antigen.

 Immunization was carried out four times with an interval of 2 weeks. The drugs were injected intramuscularly in a volume of 0.1 ml into the limb.

Blood sampling was performed by decapitation according to the following scheme:
1) a week after the second injection;
2) a week after the third injection;
3) 15 days after the fourth injection.

 The antibody titer was determined in a hemagglutination reaction (RGA) according to the generally accepted method.

 Diagnostic for RGA was prepared by the following method. To 3.2 ml of distilled water, 0.32 ml of BSA was added at a concentration of 1 mg / ml, 0.1 ml of a 0.25% solution of glutaraldehyde and 0.1 ml of a 4% solution of red blood cells, stirred and left at room temperature for 1 hour ( Fremel G., 1987).

 Data on the studies are shown in table 2.

Experiment 2
The study was carried out on sexually mature rabbits in the amount of 6 pieces of chinchilla breed. The objects were divided into three groups of 2 animals in each group and were immunized according to the following scheme (table 3).

 Immunization was performed twice with an interval of 2 weeks. After the second immunization, a boost was carried out. The drugs were injected intramuscularly in a volume of 0.1 ml into the limb.

 Freund's complete adjuvant was administered subcutaneously at four points along the spinal column.

Blood sampling was performed using the ear vein method according to the following scheme:
1) a week after the first injection;
2) a week after the second injection;
3) 7 days after the boost.

 The antibody titer was determined in a hemagglutination reaction (RGA) according to the generally accepted method.

 Diagnostic for RGA was prepared by the following method. To 3.2 ml of distilled water was added 0.32 ml of BSA at a concentration of 1 mg / ml, 0.1 ml of a 0.25% solution of glutaraldehyde and 0.1 ml of a 4% solution of red blood cells, stirred and left at room temperature for 1 hour.

 Data on the studies are shown in table 4.

Experiment 3
Vitamins A and E were mixed with a surfactant at a temperature of 30-50 ^o C. Sodium ascarbate having a pH of 6.7-7.2 was added to the resulting solution with a calculated amount of water. After complete mixing, a pH control is carried out. And the resulting preparation is mixed with the antigen. The dosage form is sterilized by membrane filtration. The study was carried out on sexually mature rabbits in the amount of 6 pieces of chinchilla breed. The objects were divided into three groups of 2 animals in each group and were immunized according to the following scheme (table 5).

 Immunization was performed twice with an interval of 2 weeks. After the second immunization, re-immunization was carried out. The drugs were administered intramuscularly.

Blood sampling was carried out from the ear vein according to the following scheme:
one week after the first immunization;
one week after the second immunization;
a week after reimmunization.

 Y. Pseudotuberculosis antigen was obtained according to a generally accepted technique (sonication followed by precipitation of the components of the cell wall).

 The immunological activity of the obtained antigen was determined by immunodiffusion with polyvalent yerseniotic serum. The concentration of the resulting AH led to a value of 100 μg / ml protein.

 A solution of colloidal gold was prepared according to a standard method.

The concentration of particles of gold 10 ¹² per 1 ml of solution. The drug AG was mixed with a solution of colloidal gold, as indicated in Table 3. The amount (titer) of antibodies was determined in the hemagglutination reaction (RGA) according to the standard method.

 The results of the titer of formation of antibodies to hypertension are shown in Table 6.

 Thus, the data presented show the effectiveness of the micellar form of surfactants as an adjuvant.

Claims (2)

1. Adjuvant for biological products containing a base, distilled water, a preservative, characterized in that the base adjuvant contains neoinogenic surfactants in the following ratio of components, wt.%:
Nonionic surfactants - 5-30
Preservative - 0.05-5
Distilled Water - Up to 100
2. The adjuvant for biological products according to claim 1, characterized in that it further comprises an immunomodulator in the form of fat and / or water soluble vitamins selected from the group consisting of vitamins A, E, C, or a combination thereof, in an amount of from 3 to 18 wt.%. 3. The adjuvant for biological products according to claim 2, characterized in that it contains vitamin A, E, C as fat and / or water soluble vitamins in the following ratio, wt.%:
A - 3
E - 5
C - 10
4. The adjuvant for biological products according to claim 1, characterized in that it further comprises a co-solvent in an amount of 1-25 wt.%.

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