Serum immunobiological preparation for emergency anthrax prevention and treatment

FIELD: medicine.

SUBSTANCE: substance of the invention involves sterile liquid or dry specifically active F(ab')2-fragments of anti-anthrax antibodies containing (35±5) mg·cm3 of protein and at least 96% of F(ab')2-fragments of antibodies recovered from liquid equine anti-anthrax immunoglobulin prepared of blood serum of horses preliminary immunised with strains B anthracis "СТИ"-1 and Ichtiman, and also a anthrax toxin produced by the Kohn's spirit deposition method.

EFFECT: lower reactogenicity and improved immunogenicity.

1 tbl

 

The invention relates to medical immunobiological preparations intended for emergency prophylaxis and treatment of anthrax in humans.

The famous "Globulin protivoseborainey horse liquid" (the Fund 42-3770-99, the Fund 42-0095-4537-03). The drug is a 8-10% solution immunoactive gamma - and beta-globulin fractions of hyperimmune serum against anthrax horses and is used for emergency prophylaxis and treatment of severe forms of anthrax (Handbook on the application of bacterial and viral agents. Ed. Sgjaoyvu, Raw. - M, Medicine, 1975. - 223 C.).

The closest to the proposed solution is "Globulin protivoseborainey horse dry" (Patent RF №2214836)obtained from 10-11% (protein) solution of immunoglobulin gamma and beta globulin fractions isolated from the proteins of the blood serum of immunized horses.

These drugs are whey heterogeneous drugs and are used for prophylaxis and treatment of anthrax, they have a specific precipitating, agglutinins and Coccinellidae activity.

The reason for preventing the wide use of known drug - high reactogenicity. Proteins included in its composition, due to its heterogeneity have expressed the Antiga is related to human properties. The introduction of this drug into the patient may be accompanied by signs of sensitization until the development of serum sickness and anaphylactic shock.

One of the ways to reduce the risk of complications and improve the efficiency of the use of immunoglobulins in the treatment and specific emergency prevention is the development of low molecular weight whey immunologically active drugs that can be used in large quantities for the rapid achievement of therapeutic effect at a significantly lower probability of developing complications. Improving the efficiency of specific serum drugs is possible by obtaining F(AB')2fragments of immunoglobulin protivovirusnogo horse, which, in contrast to known drugs do not contain the Fc-fragments of beta - and alpha-globulins.

The present invention is the production of finished dosage form of the drug for emergency prophylaxis and treatment of anthrax, low reactogenicity and high efficiency in comparison with the known compounds.

This result in the implementation of the invention is achieved in that the preparation contains F(AB')2fragments of immunoglobulin protivovirusnogo horse, preserving the specific activity and the antibodies (precipitating, agglutinating, cocinatermalium). The drug is a sterile specifically active fraction F(AB')2fragments of immunoglobulin protivovirusnogo horse containing (35±5) mg·cm-3protein, not less than 96% F(AB')2-fragments of the antibodies having an index of immunity in the test passive protection of mice not less than 4.0, selected from immunoglobulin protivovirusnogo horse liquid prepared from the serum of horses, previously immunized with strains of B. STI-1 and Ihtiman, as well as anthrax toxin, obtained by alcoholic precipitation at Stake.

The immune serum of horses with activity in reaction diffusion precipitation with anthrax cytoplasmic antigen (RDP with LCA) is not less than 1:20 (title) is used for preparation of immunoglobulin protivovirusnogo horse, whose activity in the RDP with the ATC shall be not less than 1:60 (title).

To highlight protivoseborainey F(AB')2fragments as a source of raw materials use of specific immunoglobulin or its intermediate product obtained alcohol by the method according Stake of hyperimmune serum of horses. There will be an additional purification of immunoglobulin from the ballast β1 and β2 globulin protein fractions that represent the most macrom the molecular, labile and antigenic portion of serum globulins. The secretion of immunoglobulins G is carried out with ammonium sulfate at a concentration of 31-35% saturation and neutral pH value. Is the hydrolysis of immunoglobulin G at a concentration of pepsin, equal to 2% at a temperature of 33 to 37°C in an acidic environment. Purification of F(AB')2 fragments of immunoglobulin from nonspecific proteins carried out by gel-filtration on ultragel ACA-34 and sephacryl SH-200. Concentration - using vysalivaniya ammonium sulfate. Re-suspension is carried out in 0.9% sodium chloride solution to a protein concentration of 30-40 mg·cm-3, dialysis until a negative reaction to the ions NH4+. Sterilization concentrated F(AB')2fragments protivovirusnogo immunoglobulin spend on installing sterilizing filtration through a cellulose acetate membrane with a pore size of 0.2 μm under a pressure of 0.2 MPa.

Drying of the drug, the dye-sublimation units (MASS 25, MASS-5, UL-1,0 and others) at the temperature of the material minus 35-45°C, With a gradient of not more than 3°C per hour, the final drying of the material is conducted at a temperature of (30±1)°C for (5±1) C. the Composition of the medium drying: concentration of sucrose (7,0±0,1)%, the concentration poliglyukina (1,0±0,1)%.

F(AB')2fragments of immunoglobulin protivovirusnogo horse liquid and dry designed for the treatment and prevention when the change anthrax. The drug is much less reactive to the human body compared to globulin protivoseborainey horse due to a significant reduction (3 times for protein) volume injected a foreign protein and the absence of composed the most reactive protein fraction (Fc-fragments), which allows its introduction in large compared with the nearest similar doses. The drug effectively and more safely conduct emergency prophylaxis and treatment of anthrax infection of varying severity. F(AB')2fragments of immunoglobulin retain precipitating, agglutinating and cocinatermalium activity of the antibodies that do not bind complement, not recorded in the kidneys due to the absence of Fc fragments, forming a connection with the heterologous tissues. In addition to the antitoxic action of F(AB')2fragments of immunoglobulin inhibit the germination of spores, and the process of capsulorraphy. According to this feature they are superior to similar properties of therapeutic immunoglobulins in 1,5-2,0 times. The drug has a higher specific activity and low reactogenicity due to a significant decrease in the share of ballast impurities and molecular weight of the introduced "alien" protein.

Our recommended value of human-dose F(AB')2fragments for the prevention of anthrax 40-50 cm 3(1.2-2.0 g protein), for therapeutic purposes 60-100 cm3(1.8 to 4.0 g of protein). These doses for protein in three and more times lower than for intramuscular use globulin protivovirusnogo horse dry (RF Patent No. 2214836), which significantly reduces the reactogenicity F(AB')2-fragments.

Physico-chemical and biological properties of the drug, F(AB')2fragments of immunoglobulin protivovirusnogo horse presented in the table.

Physico-chemical and biological properties of the drug, F(AB')2fragments of immunoglobulin protivovirusnogo horse
Name of indicator unit of measureRequirements of the project FundExperimental sample preparation
123456
Transparency unit opt. square0,05, no more than0,010,005 0,0050,0220,0260,03
Chroma, a unit of the protected area. square0,15, no more than0,0010,0150,001being 0.0360,0430,075
The protein content, mg·cm-330, not less than303540303730
Electrophoretic homogeneity is the content of F(ab')2fragments %96, not less than989798969798
pH, pH units6,6-7,06,8 6,86,67,07,07,0
SterilitySterileSterile
ProgenoteNephrogenicApirogenny
ToxicityNon-toxicNon-toxic
Activity in the reaction diffusion precipitation with anthrax cytoplasmic antigen titer1:8, not less than1:161:161:81:321:161:32
Index immunity test passive protection of mice, the river. units4,0, not less than5,05,05,05,0 5,04,0

Serum immunobiological preparation for emergency prophylaxis and treatment of anthrax, representing a sterile liquid or dry specifically active F(AB')2fragments protivoseborainey antibodies containing (35±5) mg·cm-3protein, not less than 96% F(ab')2-fragments of the antibodies having an index of immunity in the test passive protection of mice not less than 4.0, selected from immunoglobulin protivovirusnogo horse liquid prepared from the serum of horses, previously immunized with strains of B. STI-1 and Ihtiman, as well as anthrax toxin, obtained by alcoholic precipitation at Stake.



 

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