A drug for prevention and treatment of diphtheria and method thereof

 

(57) Abstract:

The invention relates to medicine, in particular immunology, and can be used for immunoprophylaxis and immunotherapy of diphtheria. The product is a lyophilized anoreceptive fraction F(ab)2fragments of IgG horse, isolated from fermented hyperimmune horse serum using immunosorbent assay containing as a ligand purified concentrated diphtheria toxoid. Immunoaffinity cleaning can obtain the product with the activity of diphtheria antibodies at least 2500 IU/ml, a protein content of 3-4%. For the preparation of diphtheria drug use hyperimmune horse serum, for what it ferment pepsin, perform salt fractionation of proteins and thermodenaturation ballast protein. The selection of the target product from fermented hyperimmune serum is performed with the use of specific sorption. The technical result consists in obtaining highly purified diphtheria drug with reduced reactogenicity. 2 S. and 1 C.p. f-crystals, 3 tab., 2 Il.

The invention relates to medicine, in particular immunology, and can be ipoi is intramuscular diphtheria serum (PDS), obtained from the blood of horses, hyperimmunization with diphtheria toxoid containing specific antibodies, neutralizing the toxins of diphtheria bacteria. 1 ml serum contains not less than 1500 international toxic units of activity (IU). Being an effective method for the treatment of diphtheria, MPD has a number of disadvantages: the introduction of significant amounts of foreign protein leads to the possibility of anaphylactic reactions and serum sickness (Favorov L. A. and others Diphtheria. - M.: Medicine, 1988. - S. 187-192).

Known for the preparation of immunoglobulin diphtheria human, plasma-derived donors vaccinated with diphtheria toxoid. 1 ml of the preparation contains from 5 to 50 IU of diphtheria antibodies. Prophylactic dose of 300 IU of intramuscular introduction, treatment and from 1200 to 20000 IU (Aleshin C. A., Zatsepin Y. K., Bally A., and others Receiving diphtheria immunoglobulin. // The role of vaccines in modern medicine. - Ufa, 1995. - 1 o'clock. - S. 100-102).

However, the issue of diphtheria homologous immunoglobulin is limited by insufficient resources and weak immunization schemes. More limited is acity, HIV infection, new T - and B-lymphotropic viruses, diagnostics are still difficult) (raikher L. I. , raikher And. And. Ofinnocence xenogenic antibodies. Prospects, problems. // The role of vaccines in modern medicine. - Ufa, 1995. - 2 hours. - S. 86-91).

In addition, for preparation of one batch of immunoglobulin diphtheria person you want blood not less than 1000 donors, so it is a mixture of molecules of different allotype, the introduction of which may lead to the development of allergic reactions in patients (Picky Century. And., Adrianova N. Century , Artamonova A. C. Allergic diseases. - M.: Medicine. - 1991. - S. 176).

Closest to the proposed invention is a method for heterogeneous serum preparation, which includes the processing of whey with rivanol, pepsin, aluminum hydroxide, followed by ultrafiltration and sterile filtration (patent N 2062617, class A 61 K 35/16, 39/00, 1996).

However, this method does not allow to obtain products with a high degree of purification: the final product is a complex of F(ab)2fragments of antibodies of different specificity, and antibody (antivenoms) protiva this antitoxic serum remain overloaded ballast proteins and in some cases may cause anaphylactic reactions of varying severity on the part of the recipient. In addition, drugs contaminated group substances in the blood (GVK), which may also cause complications with the introduction of serum.

The technical result of the invention is to obtain highly purified diphtheria drug with reduced reactogenicity of heterogeneous materials. A drug for prevention and treatment of diphtheria is a lyophilized form.

The problem is solved by obtaining the drug for the prevention and treatment of diphtheria from fermented hyperimmune horse serum using immunoaffinity cleanup.

The invention consists in the following. A drug for prevention and treatment of diphtheria is a lyophilized anoreceptive fraction F(ab)2fragments of IgG horse, isolated from fermented hyperimmune horse serum using immunosorbent assay containing as a ligand purified concentrated diphtheria toxoid. Immunoaffinity cleaning allows to obtain a highly purified drug for the prevention and treatment of diphtheria with the activity of diphtheria antibodies at least 2500 IU/ml and a protein content of 3-4%.

The resulting preparation is m the Arat for the prevention and treatment of diphtheria is anoreceptive: in response double immunodiffusion with diphtheria toxoid forms a single line of precipitation, while serum (prototype) - 2-3 lines (Fig. 2).

Characteristics of the drugs obtained by known methods and proposed, are presented in table. 1-3.

High degree of purification and the lack of GVK reduce reactogenicity of a drug for prevention and treatment of diphtheria, derived from heterogeneous sources (table. 1, 2), while it has a high protective properties: minimum dose of antitoxin in the blood sera of 0.03-0,125 IU/ml, corresponding protective titer diphtheria, protects 100% of the animals from the introduction of 1.8 to 5.6 LD50diphtheria toxin (table. 3). Freeze drying allows to obtain highly active dry dosed medications, suitable for long term storage.

The implementation of the method.

For the preparation of a drug for prevention and treatment of diphtheria use hyperimmune horse serum, for what it ferment pepsin, perform salt fractionation of proteins and thermodenaturation ballast protein. The selection of the target product from fermented hyperimmune serum is performed with the use of specific immunosorbent assay containing as a ligand purified concentrated gift the STV molecular weight of 100 KD. Sterilizing filtration is performed on membranes with a pore diameter of 0.22 μm.

Example

1. Fermentas hyperimmune serum.

30 l hyperimmune horse serum is loaded into the reactor and diluted with 60 l of pyrogen-free distilled water to a content of 3% protein. Contribute with stirring 243 g of pepsin dissolved in a small amount of acidified distilled water. The proteolysis is carried out at a pH of 3.2 at room temperature for 1 h and 1 h at pH of 4.2. (pH adjusted to the desired value of 2.5 mol/l solution of hydrochloric acid or 2.5 mol/l sodium hydroxide solution).

At the end of the 2nd hour of proteolysis in the reactor through the loading hatch add 14.5% of ammonium sulfate (13 kg), pH adjusted to a value of 4.3-4,33. The mixture is heated to 56oC and kept at this temperature for 45 minutes. After that fermentat released from ballast protein by filtration through mickalene cassette filters. Sediment ballast proteins cast, but the clear filtrate containing proteins with antioxidant activity, collect and bring it pH to 7.0 to 7.2.

2. Preparation of monospecific immunosorbent assay.

To obtain immunosorbent assay uses the standard drug lambropoulou sexin activity 450 Lf/ml 200 g dry sepharose pour two liters of 0.001 mol/l hydrochloric acid for 30 minutes and washed from preservatives the same solution. 1.2 l of diphtheria toxoid combined with sediment turgid sepharose (900 ml), mixed well and incubated at a temperature of 4oC for 24 hours. Then determine the volume of the supernatant liquid (V1and the title toxoid (T1in response flocculation. If the title does not exceed 10 Lf/ml, decanted drop, otherwise sorption continue. The complex formed of sepharose - toxoid washed from unbound protein sterile pyrogen-free carbonate-bicarbonate buffer solution pH to 9.0 under control spectrophotometry. The last portion of the wash water after the establishment of neutral pH test pyrogen according to the standard technique.

In the absence of pyrogens in the lavage free active groups lambropoulou separate block two liters of 1 mol/l solution of ethanolamine pH 9.0 in for two hours in a darkened place with constant stirring.

Immunosorbent washed three times alternating sterile pyrogen-free buffer solutions: 2 l acetate pH 4,0; 2 l boric-borate pH 8.0. The washing water (VSchityvat by the formula

< / BR>
where A is the title immunosorbent assay;

V T is the volume and titer toxoid;

V1, T1- volume and titer of the supernatant liquid;

V2, T2- volume and titer of wash water;

C - volume immunosorbent assay.

< / BR>
So, get 900 ml suspension immunosorbent assay with titres 588 Lf/ml

3. Selection anoreceptive fraction F(ab)2fragments of IgG horse, the concentration of the drug.

To highlight the F(ab)2fragments of IgG horse use beskonechny "Batch"method. All of the used capacity and the solutions must be sterile and pyrogen-free. Sorbent and fermented antitoxic serum activity 110 IU/ml are combined in amounts equivalent to a titer of

< / BR>
where Vf, Tf- volume and titer of fermentata,

VI. S., TI. S.- volume and titer immunosorbent assay.

< / BR>
I.e. one portion immunosorbent assay enough to exhaustion 4.8 l fermented diphtheria antitoxic serum.

Sorption of specific antibodies was performed at room temperature for 1 h, pH 7.0. The sorbent is washed from unbound proteins sterile pyrogen-free buffered saline (SFR) pH 7.0 is, okislennye to pH 2.2 to 2.4 to 1 mol/l hydrochloric acid.

Exposure immunosorbent assay in eluent is 10-15 minutes. Specific protein sequentially elute portions SFR pH 2,4: 1,5-1,0-0,5 L. Select eluate with a protein content not less than 0.01%, unite and bring the pH to 6.8 to 7.2. United eluate tested for pyrogen according to the standard technique.

Immunosorbent washed from protein SFR (pH 7.0) and stored in sterile pyrogen-free boric-borate buffer solution (pH 8.0) at a temperature of 4oC. immunosorbent use repeatedly (20 times).

Get 90 l eluates with the contents of a specific protein 0,095-0,1%. The concentration of the eluates carried out by ultrafiltration installation AR-2 with hollow fibers brand SPM-100 under pressure 0,06 0,02 MPa. The concentration process continued until the content of protein in the concentrate within 3-4%.

Get 3 liters of concentrated ofinnocence F(ab)2fragments of IgG horse with the activity of 2500 IU/ml and protein-3%. Purified concentrated antitoxin lighten using serial filtration through membranes with pore diameter of from 0.8 to 0.65-0.45 μm. Sterilizing filtration is performed on membranes with a pore diameter of 0.22 μm. Sterile drug profile the ohms for the prevention and treatment of diphtheria is placed in the receiving cassette and put on a freeze in low temperature counter apparatus TG-50, providing a freezing temperature not less than minus 38oC. With forced ventilation the product is frozen for at least 18 h, without forced ventilation of at least 20 hours Download of the product in sublimator is performed upon reaching the condenser temperature not higher than minus 50oC, shelf no higher than minus 15oC. After which sublimator vacuumized. The point of thawing of the drug minus 33-36oC. When the product temperature not higher than minus 38oC and stabilization of vacuum of not more than 9 PA start heating shelves. One hour after heating load in 0oC and then for 5oC per hour. The final temperature of the heating shelves 34-40oC. After incubation of the drug at zero temperature for 20 h drying is complete.

Thus, the proposed method allows to obtain the drug for prevention and treatment of diphtheria with reduced reactogenicity with high protective properties, in lyophilized form.

1. A drug for prevention and treatment of diphtheria containing F(ab)2-fragments of IgG horses, characterized in that it is anoreceptive fraction F(ab)2fragments of IgG horse with activity about the sodium with a pH of 6.8 - 7,2 in the following ratio of ingredients:

Anoreceptive fraction F(ab)2fragments of IgG horse activity of diphtheria antibodies at least 2500 IU/ml, 3 to 4 wt.%

0.9% sodium chloride solution with a pH of 6.8 to 7.2 To 1 ml

2. A drug for prevention and treatment of diphtheria under item 1, characterized in that it is a lyophilized form.

3. The method of producing drug for the prevention and treatment of diphtheria, including fermentors hyperimmune horse serum pepsin, characterized in that after the fermentation is performed with saline fractionation and thermodenaturation of serum proteins, the selection of the target product is carried out using specific immunosorbent assay containing as a ligand purified concentrated diphtheria toxoid, followed by concentration by ultrafiltration on membranes with a threshold detention substances on the molecular mass of 100 KD, sterilizing filtration on membranes with a pore diameter of 0.22 μm.

 

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FIELD: medicine, ophthalmology.

SUBSTANCE: one should apply an autohemocomponent preparation being supernatant liquid of patient's autoblood at increased serotonin content obtained due to irreversible thrombocytic aggregation due to the impact of 0.5 mg ATP per 1.0 ml plasma followed by a 30-min-long centrifuging at the rate of 1000, 2000 and 3000 rot./min for 20, 7 and 3 min, correspondingly. In case of no exudative phenomena on patient's eye bottom the obtained preparation should introduced at the quantity of 7-10 ml once in 48 h for 1 mo (totally, 15 injections). In case of exudative-hemorrhagic phenomena it should be introduced parabulbarly at the volume of 0.5 ml and parenterally - 7.0-10.0 ml once in 48 h for 1 mo per 15 injections, correspondingly. The preparation enables to improve visual functions due to decreased tissue hypoxia and normalization of microcirculation in visual analyzer.

EFFECT: higher efficiency of therapy.

2 cl, 7 dwg, 2 ex

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