RU2206337C1 - Medicinal preparation for treatment of muscle dystonia and method for its preparing - Google Patents

Abstract

FIELD: medicine, microbiology, pharmacy. SUBSTANCE: medicinal preparation comprises botulinic toxin isolated from culture Clostridium botulinum type A, strain = 98, as a liquid substance dissolved mainly in 0.87% 0.04 M solution of sodium chloride, pH 4.9-6.9. Method for preparing medicinal preparation involves preparing botulinic toxin type A from culture Clostridium botulinum, strain = 98, by its culturing at temperature 29-30 C for 3-6 days in nutrient medium. Then dry ammonium sulfate is added slowly to cultural fluid to form precipitate, prepared suspension is centrifuged to isolate precipitate and isolated liquid phase is extracted and subjected for purification. Process of purification of liquid substance is carried out for two stages where at the first step nickel-base metallosorbent is used mainly and at the second step ion-exchange sorbent at pH 6.1-6.9 is used. Sterilization of purified substance is carried out by membrane filtration. Invention provides preparing the preparation no losing activity in liquid form. EFFECT: improved preparing method, valuable medicinal properties of preparation. 3 cl, 1 ex

Description

 The invention relates to medicine and relates to the creation of a drug intended for the treatment of human muscular dystonia, such as strabismus, torticollis, involuntary blinking, etc.

Known from Schantz EJ, Johnson EA Properties and use of botulinum toxin and other neurotoxins in medicine. Microbial.Rev., 1992, 56, 80-99 drug and its preparation on the basis of botulinum B, including cultivation of a strain of Clostridium botulinum type B, sedimentation of the toxin together with bacterial cells at pH 3.7, extraction with 1 M NaCl, precipitation with ethanol (final concentration 15%), dissolution in phosphate buffer and subsequent crystallization during dialysis against 0.9 M ammonium sulfate. After recrystallization, the botulinum toxin is diluted in 0.15 M NaCl to a concentration of 100Ld ₅₀ / ml, human serum albumin is added to a concentration of 500 μg / ml, sterilized and lyophilized.

 The well-known drug “VOTOX” is used to treat muscular dystonia.

 The disadvantage of this drug is its relatively low activity due to incomplete purification of the latter on the one hand, and on the other, the loss of activity as a result of the lyophilization process.

 The closest in technical essence and the achieved effect are known from Das Gupta B.R., Sathyamoorthy V. Purification and amino acid composition of type A botulinum neurotoxin. Toxicon, 1984, 32, 415-434, a medicament for the treatment of muscular dystonia, containing botulinum toxin type A, isolated from a culture of Clostridium botulinum albumin and a method for producing a medicament for the treatment of muscular dystonia, including cultivating the strain on culture medium, sedimentation, extraction, purification using DEAE cellulose, sterilization and lyophilization.

 The disadvantages of the known drug for the treatment of muscular dystonia and the method of its production is that it contains not only botulinum toxin, which is resistant to proteolytic degradation in the body, but also botulinum neurotoxin, which is very sensitive to inactivation by proteolytic enzymes, while it does not retain its activity during storage in liquid form and therefore requires the inclusion of a lyophilization step.

 The objective of the invention is to provide the possibility of obtaining a medicinal product for the treatment of muscular dystonia containing botulinum toxin type A, which does not lose its activity when stored in liquid form.

 To solve this problem, a group of inventions is proposed, united by a common inventive concept.

A medicament for treating muscular dystonia is proposed containing botulinum toxin type A isolated from a culture of Clostridium botulinum albumin, while botulinum toxin isolated from a culture of Clostridium botulinum type A strain 98 in the form of a liquid substance dissolved mainly in 0.87% 0.05 M a solution of sodium chloride with a pH of 4.9-6.9 in the following ratio of components in mg / ml:
Sodium chloride - 0.8-1.0
10% albumin solution - 0.6-0.9
Substance of botulinum toxin - 0.000001-0.000005
A method of obtaining a medicament for the treatment of muscular dystonia isolated from a culture of Clostridium botulinum type A, including culturing the strain on a nutrient medium, sedimentation, extraction, purification and sterilization, botulinum toxin type A, obtained from the culture of Clostridium botulinum strain 98, the cultivation of which is carried out at a temperature 29-33 ^o C for 3-6 days, and then carried out in two phases of liquid cleaning substance, the first of which is used metallocorbent, preferably based on nickel, and the second stage is used onoobmenny adsorbent at pH 6.1-6.9, followed by sterilization by membrane filtration.

Strain 98 is grown on a nutrient medium, the following composition in g / l:
Trypton - 25-33
Yeast Extract - 25-33
Thioglycolate - 0.2-0.6
Distilled Water - Up to 1 L
Glucose - 4-6
2N NaOH - To pH 7.1-7.5
For the preparation of seed using a frozen uterine culture of strain A98 Clostridium botulinum. A uterine culture in the amount of 0.5 ml is seeded on tryptone-yeast medium, poured into 50 ml in 100 ml vials. The culture is grown at a temperature of 30 ^o C for 3-5 days. Inoculum can be used for inoculation for 6 months, which is stored at 4 ^° C. From vials with a uterine culture, inoculation on TD medium is performed to obtain a native toxin: 2.5 ml of inoculum is introduced into 2.5 L of sterile tryptone-yeast medium material and growing the culture produced in a thermostat at a temperature of 30 ^o C for 6 days.

Then determine the toxicity of the culture fluid. 2 samples of culture fluid of 1.0 ml each are taken. In both samples, the biological activity in DLM / ml was determined by titration on outbred white mice. For this, the drug is diluted in gelatin-phosphate buffer in accordance with the intended titer. For example, to determine the toxin titer in the range of 5 • 10 ⁵ DLM / ml, it is diluted sequentially with a tenfold interval to 1 • 10 ⁵ and then 2 • 10 ⁵ , 4 • 10 ⁵ . From each dilution, 0.5 ml of the toxin is injected intraperitoneally with two syringes to two mice weighing 16-30 g, starting with a larger dilution. The maximum dilution of the toxin that caused the death of mice within 4 days, contains 1 DLM. The biological activity of the drug is equal to the dilution value, multiplied by 2, and is expressed in DLM / ml.

Type specificity is also determined at this stage. For this, the culture fluid, after trypsin activation, is diluted to a concentration of 100,000 DLM / ml and a neutralization reaction is performed on white mice with diagnostic anti-botulinum sera A, B and E. In 3 tubes, 1 ml of the prepared dilution of culture fluid with a concentration of 100,000 DLM / ml is poured. In the first test tube add 1 ml of diagnostic serum type A, in the second - type B, in the third - type E (all sera with a concentration of at least 100 IU / ml). The mixture is stirred and incubated for 30 minutes at room temperature. Then, 0.5 ml of a mixture of toxin with serum is taken from each tube and two mice are injected intraperitoneally with different syringes. Within a few hours, 4 mice that were injected with a mixture of toxin with serum types B and E should die, and mice that received a mixture of toxin with serum type A should survive. In this case, the toxin is considered type-specific. The culture fluid is used in further work if the toxin contained in it is strictly type-specific (type A) and the toxicity of 1 ml of the solution is not less than (4-7) • 10 ⁵ DLM / ml.

To the resulting volume of culture fluid in the bottle add 3N H ₂ SO ₄ to a pH of 3.5-4.0. PH control is carried out using a paper pH indicator in the first stages, and then finally on the pH meter. The mixture is left for 2-18 hours at room temperature to form a precipitate. The culture fluid is centrifuged at 6000 rpm for 10 min at 4 ^o C. The supernatant is drained, and the precipitate is used for further work.

For the extraction of the toxin, 0.2 M acetate buffer pH 6.5-6.7 containing 1 M NaCl is used. An extraction buffer is added to the precipitate (1/20 of the initial volume, for example, 125 ml of buffer is added to the precipitate from 2.5 L of culture liquid), suspended by pipetting thoroughly and extracted for 1 hour with constant stirring. The suspension is centrifuged for 10 minutes at 4 ^° C. The supernatant is poured into a clean beaker and the pellet is discarded. At this stage, 120-135 ml of the preparation with activity from 6 • 10 ⁶ to 1.2 • 10 ⁷ DLM / ml is obtained.

 For each liter of supernatant, 313 g of dry ammonium sulfate is slowly added, thoroughly mixing with a glass rod.

 To form a precipitate, the solution is left for 17-24 hours at a temperature of 5-3%, after which the preparation can be used for further work.

The toxin preparation in a solution of ammonium sulfate can be stored at 3-5 ^o C for 2 years.

The suspension is centrifuged at 15,000 rpm for 10 min at 4 ^o C. The supernatant is drained, and the precipitate is used in further work.

40 ml of 50 mM phosphate buffer pH 6.8 is added to the precipitate from 60 ml of toxin, thoroughly mixed with a glass rod until the precipitate is completely dissolved and adjusted to 50 ml with 50 mm phosphate buffer pH 6.8. The drug is centrifuged at 15,000 rpm for 10 minutes at 4 ^° C. The supernatant is poured into a clean beaker and the pellet is discarded. A 1.0 x 20 cm column is filled with 5 ml of metal sorbent, mainly based on nickel, and washed with 20 ml of 50 mM phosphate buffer pH 6.8. Then, at room temperature, 50 ml of the toxin solution is applied to the sorbent column using a peristaltic pump at a rate of 1 ml / 2 min. After passing the toxin, the column is washed with 20 ml of 50 mM phosphate buffer pH 6.8.

 The toxin is eluted with 10 ml of elution buffer (0.05 M phosphate buffer pH 6.5, 0.05 M NaCl, 0.02 M EDTA). Fractions were collected in 1 ml sterile glass tubes. Fractions containing purified toxic complex are combined and used for further work.

 Low molecular weight impurities in the preparation are removed either by ion-exchange chromatography on anion exchangers, or by gel filtration. For example, a column measuring 1.0 x 20 cm (column volume 5 ml) is filled with 5 ml of DE-Sephacel granular ion-exchange sorbent and washed with 200 ml of 50 mM phosphate buffer pH 6.5. 10 ml of the toxin solution is applied to the column using a peristaltic pump at a rate of 1 ml / 1 min. After passing the toxin, the column is washed with 30 ml of 50 mM phosphate buffer pH 6.5.

 The toxin is eluted with 20 ml of 0.1 M pH 5.5 citrate-phosphate buffer. Fractions were collected in 1 ml sterile glass tubes. The fractions containing the purified toxin are combined, the volume is adjusted with 0.1 M citrate-phosphate buffer pH 5.5 to 10 ml and marked as purified botulinum toxin type A.

 The purified toxin is placed in a syringe and sterilized by filtration through a 0.22 μ filter nozzle into a sterile glass tube in a laminar box.

At this stage, the drug determines the protein, activity in DLM in mice, activity in LD ₅₀ and specific activity.

 The protein is determined spectroscopically at 280 nm, based on the fact that the concentration of purified botulinum toxin type A 0.1% gives an absorption of 1.65 at 280 nm. The protein concentration of purified botulinum toxin is 0.2-0.3 mg / ml.

The activity of the drug in DLM is determined in mice, as described above. The activity of the purified botulinum toxin preparation is from 3 • 10 ⁶ to 1 • 10 ⁷ DLM / ml.

The activity of the drug in LD ₅₀ is determined in mice as follows: 1 ml of the drug is diluted to a concentration of 1000 DLM / ml and LD _{50 is} determined in it. Activity is determined by examining the toxicity of the drug in mice. For this purpose, gelatin-phosphate buffer pH 6.5 and outbred mice, the body weight of which is in the range from 17 to 23 g, are used.

To determine Ld ₅₀ use 2 ml of the drug. To each 1 ml of the drug add 1.8 ml of saline and then make the following dilutions:
dilution 0: 4.4 ml of gelatin-phosphate buffer is added to 2.2 ml of the drug;
dilution 1: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 0;
dilution 2: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 1;
dilution 3: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 2;
dilution 4: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 3;
dilution 5: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 4;
dilution 6: 1.45 ml of gelatin-phosphate buffer is added to 4.4 ml from dilution 5.

 Each animal is injected with 0.1 ml of each dilution intraperitoneally of each mouse (in a group of at least 6 animals). The number of dead animals is recorded within 96 hours after administration of the drug.

Calculation: LD ₅₀ and 95% confidence interval are calculated using the Ashmarin-Kerber method under the following conditions:
a) all groups must have the same number of animals,
b) in the group to which the most concentrated drug was administered, 100% death of animals should be observed,
c) in the group to which the least concentrated drug was administered, the death of animals should not be observed,
d) all solutions should have a constant dilution ratio.

Calculation of LD _{50 is} carried out according to the formula
m = Ld (Σpj-0.5),
where LD ₅₀ - breeding, in which the death of 50% of the animals;
m is the logarithm of LD ₅₀ (log LD ₅₀ );
L is the logarithm of the dilution of the highest concentration of the drug in the input series;
d is the log of the dilution factor;
pj is the proportion of animal death in j breeding;
Σpj is the sum of the shares of the death of animals.

где Σ[pj(1-pj)] - сумма долей гибели животных при j разведении, умноженная на (1- сумма долей гибели животных при j разведении);
n - число животных на группу для испытуемой дозы.The standard error is calculated using the following formula:

where Σ [pj (1-pj)] is the sum of the shares of the death of animals at j breeding, multiplied by (1 is the sum of the shares of death of animals at j breeding);
n is the number of animals per group for the test dose.

95% confidence interval is calculated by the following formula:
Upper bound = M + (1.96 x standard error)
Lower bound = M - (1.96 x standard error)
Results are converted to LD ₅₀ units using antilogarithm tables. Due to the fact that only 0.1 ml of solution is administered to each mouse, the values of the LD ₅₀ units are multiplied by 10 to express the results in units of LD ₅₀ per 1 ml.

The activity of the purified botulinum toxin preparation is from 9 • 10 ⁶ to 3 • 10 ⁷ LD ₅₀ / ml.

The specific activity of the drug is calculated by the formula:
the amount of LD ₅₀ / ml
Specific Activity (LD ₅₀ / mg)
The specific activity of purified botulinum toxin preparations is from 3 • 10 ⁷ to 1 • 10 ⁸ LD ₅₀ / mg.

 For the preparation of the final drug, use "Isotonic 0.9% sodium chloride solution for injection" or 0.05 M citrate-phosphate buffer pH 5.5 and "Albumin solution 10% intravenously". For this purpose, in sterile conditions, an albumin solution is added to a sodium chloride solution or buffer to a final albumin concentration of 500 μg / ml: 6 ml of an albumin solution is added to 1194 ml of a sodium chloride solution. Under sterile conditions, a calculated amount of the substance is added to 1200 ml of the buffer solution so that the final activity of the batch volume is 100 u / ml.

где Х - количество мл субстанции;
V_b - объем серии препарата (мл);
V_f - объем препарата во флаконе;
U_e - количество единиц во флаконе;
U_s - количество единиц/мл в V_b.The dilution factor is calculated by the following formula:

where X is the number of ml of substance;
V _b is the volume of the series of the drug (ml);
V _f is the volume of the drug in the vial;
U _e is the number of units in the bottle;
U _s is the number of units / ml in V _b .

х=0,545 мл.Calculation example: if the volume of a series of a preparation is 1200 ml, the volume of a preparation in a bottle is 1 ml, the number of units in a bottle is 100, and the substance solution contains 2.2 • 10 ⁶ units / ml, then

x = 0.545 ml.

 The resulting final product is poured into 1 ml into 5 ml vials.

 Operations are carried out under sterile conditions in a laminar box.

 After filling, the vials are closed with a sterile stopper and plugged with foil.

The resulting preparation retains its activity for at least 6 months when stored at 4-8 ^o C.

 An example implementation of the method of obtaining a medicinal product for the treatment of muscular dystonia.

 The composition of the culture medium for the cultivation of strain A98 Clostridium botulinum in g / liter: tryptone - 30 g, yeast extract - 30 g, thioglycolate - 0.5 g, distilled water - up to 1 l, 2N NaOH - up to pH 7.3.

The prepared medium is autoclaved at 110 ^{° C.} for 30 minutes at 0.5 atm.

For the preparation of seed using a frozen uterine culture of strain A98 Ciostridium botulinum. A uterine culture in the amount of 0.5 ml is seeded on trypto-yeast medium, poured into 50 ml in 100 ml vials. Before each inoculation, glucose is added to each vial to a final concentration of 0.5%. The culture is grown at a temperature of 34 ^o C for 4-5 days. The seed can be used for sowing for 6 months if stored at 4 ^o C. From vials with uterine culture, inoculation on TD medium is carried out in order to obtain a native toxin.

Before use, 40% sterile glucose solution is added to the nutrient medium to a final concentration of 0.5%. In 2.5 l of sterile tryptone-yeast medium, 2.5 ml of inoculum is introduced and the culture is grown in an incubator at a temperature of 33 ^o C for 6 days.

2 samples of culture fluid of 1.0 ml each are taken. In both samples, the biological activity was determined in DLM / ml titration on outbred white mice. For this, the drug is diluted with gelatin-phosphate buffer in accordance with the intended titer. For example, to determine the titer of the toxin within 5 • 10 ⁶ DLM / ml, it is diluted sequentially with a tenfold interval to 1 • 10 ⁵ and then 2 • 10 ⁵ , 4 • 10 ⁵ . From each dilution, 0.5 ml of the toxin is injected intraperitoneally with two syringes to two mice weighing 16-30 g, starting with a larger dilution. The maximum dilution of the toxin that caused the death of mice on day 3 contains 1 DLM. The biological activity of the drug is equal to the dilution value, multiplied by 2, and is expressed in DLM / ml.

 Type-specificity determination is carried out after toxicity determination as described above.

The culture fluid is used in further work if the toxin contained in it is strictly type-specific (type A) and the toxicity of 1 ml of the solution is not less than (4-7) • 10 ⁵ DLM / ml.

To the resulting volume of culture fluid in the bottle add 3N H ₂ SO ₄ to a pH of 3.5-4.0. PH control is carried out using a paper pH indicator in the first stages, and then finally on the pH meter. The mixture is left for 2-18 hours at room temperature to form a precipitate.

Then carry out centrifugation, while before centrifugation of the precipitate, the centrifuge and the rotor are cooled to 5-7 ^o C. Prepare dishes for draining the centrifuge.

The culture fluid is placed in 500 ml glasses and centrifuged in a J2-21M / E medium speed centrifuge in a JA-12 rotor at 8000 rpm for 10 min at 4 ^° C. The supernatant is poured into a bottle and the precipitate is used for further work.

For the extraction of toxin using 0.2 M acetate buffer pH 6.5 to 6.7, containing 1 M NaCl. Prepare a 0.2 M solution of acetic acid: glacial acetic acid 1.13 ml, distilled water - up to 100 ml. Sodium acetate 3Н ₂ О is weighed in at 27.2 g, sodium chloride - 58 g, to which 4 ml of a 0.2 M solution of acetic acid and distilled water are added up to 1 liter. The pH of the buffer is controlled using a potentiometer.

 An extraction buffer is added to the precipitate (1/20 of the initial volume, for example, 125 ml of buffer is added to the precipitate from 2.5 L of culture liquid), suspended by pipetting carefully and extracted for 1 hour with constant stirring.

Before centrifugation of the precipitate, the centrifuge and the rotor are cooled to 5-7 ^o C. Prepare dishes for draining the centrifuge.

The suspension is placed in 250 ml centrifuge glasses and centrifuged in a J2-21M / E medium speed centrifuge in a JA-10 rotor at 10,000 rpm for 10 min at 4 ^° C. The supernatant is poured into a clean chemical beaker and the precipitate is discarded.

At this stage, 120-125 ml of the drug with activity from 6 • 10 ⁶ to 1.2 • 10 ⁷ DLM / ml is obtained.

 Knowing the volume of extracted toxin, a sample of ammonium sulfate is prepared at the rate of 313 g of salt per liter of liquid (50% saturation).

 For each liter of culture fluid, 313 g of dry ammonium sulfate is slowly added, thoroughly mixed with a glass rod.

To form a precipitate, the solution is left for 17-24 hours at a temperature of 5-3 ^o C, after which the drug can be used for further work.

The toxin preparation in a solution of ammonium sulfate can be stored at 3-5 ^o C for 2 years.

The suspension is centrifuged at 15,000 rpm for 10 min at 4 ^o C. The supernatant is drained, and the precipitate is used in further work.

40 ml of a 0.87% sodium chloride solution with a pH of 6.8 are added to a precipitate of 60 ml of toxin, thoroughly mixed with a glass rod until the precipitate is completely dissolved and adjusted to 50 ml with 50 mM phosphate buffer pH 6.8. The suspension is centrifuged at 15,000 rpm for 10 minutes at 4 ^° C. The supernatant is poured into a clean beaker and the precipitate is discarded.

 Further, commercial columns from Whatman (England) are used in the work. A column measuring 1.0 x 20 cm (column volume 20 ml), previously washed and mounted in a tripod, is filled with 5 ml of granular metal sorbent, mainly based on nickel, and washed with 20 ml of 50 mM phosphate buffer pH 6.8. The operation is carried out at room temperature.

 Then, 50 ml of the toxin solution is applied to the sorbent column using a peristaltic pump at a rate of 1 ml / 5 min. After passing the toxin, the column is washed with 20 ml of 50 mM phosphate buffer pH 6.8.

 The toxin is eluted with 10 ml of elution buffer (0.05 M phosphate buffer pH 6.5, 0.05 M NaCl, 0.02 M EDTA). Fractions containing purified toxic complex are collected 1 ml in sterile glass tubes, then combined and used for further work.

Then, 0.05 M phosphate starter buffer pH 6.5, an ion exchange sorbent buffer, is prepared. Stock solutions are prepared for the phosphate buffer: 0.2 M Na ₂ HPO ₄ 12H ₂ O - 71.6 g is dissolved in 1 liter of distilled water and 0.2 M NaH ₂ PO ₄ 2H ₂ O - 31.2 g is dissolved in 1 liter distilled water. 685 ml of a 0.2 M solution of NaH ₂ PO ₄ and 315 ml of a 0.2 M solution of Na ₂ HPO _{4 are} drained, the pH is controlled and diluted 4 times.

 In work commercial columns of the company Whatman (England) are used with the DE-Sephacel ion-exchange sorbent. A column measuring 1.0 x 20 cm (column volume 20 ml), previously washed and mounted in a tripod, is filled with 5 ml of DE-Sephacel granular ion-exchange sorbent and washed with 20 ml of 50 mM phosphate buffer pH 6.5. The operation is carried out at room temperature.

 Then 10 ml of the toxin solution is applied to the column using a peristaltic pump at a rate of 1 ml / 5 min. After passing the toxin, the column is washed with 30 ml of 50 mM phosphate buffer pH 6.5.

 The toxin is eluted with 20 ml of 0.1 M pH 5.5 citrate-phosphate buffer. Fractions were collected in 1 ml sterile glass tubes. Fractions containing the purified toxic complex combine the volume, adjusting with a pH of 5.5 to 0.1 ml with 0.1 M citrate-phosphate buffer, and mark as purified type A botulinum toxin.

 The purified toxin is placed in a 10 ml syringe and filtered through a 0.22 μ filter nozzle (Costar, Cambridge, NA, Cat. 8110 or Corning, NY, 1431) in a sterile glass tube in a laminar box.

 Production waste at this stage is a nozzle with a filter and a syringe, which are neutralized by pouring 6% freshly prepared solution of hydrogen peroxide for a day, followed by autoclaving.

At this stage, the protein is determined spectroscopically in the drug, activity in the DLM in mice, activity in LD ₅₀ and specific activity.

The specific activity of purified botulinum toxin drugs is from 3 • 10 ⁷ to LD ₅₀ / mg.

 For the preparation of the final drug use "Isotonic sodium chloride solution 0.9% for injection" manufactured by JSC "East", pos. East, Kirov region or 0.05 M citrate-phosphate buffer pH 5.5 and "10% solution of albumin intravenously" manufactured by GUP "Immunopreparat", Ufa. For this purpose, in sterile conditions, an albumin solution is added to the buffer to a final albumin concentration of 5 mg / ml: 6 ml of albumin solution is added to 1200 ml of the buffer solution.

где X - количество мл субстанции;
V_b - объем серии препарата (мл);
V_f - объем препарата во флаконе;
U_e - количество единиц во флаконе;
U_s - количество единиц/мл в V_b.The dilution factor is calculated by the following formula:

where X is the number of ml of substance;
V _b is the volume of the series of the drug (ml);
V _f is the volume of the drug in the vial;
U _e is the number of units in the bottle;
U _s is the number of units / ml in V _b .

х=0,0545 мл или 54,5 мкл.Calculation example: if the volume of a series of a preparation is 120 ml, the volume of a preparation in a bottle is 0.1 ml, the number of units in a bottle is 100, and the substance solution contains 2.2 • 10 ⁶ units / ml, then

x = 0.0545 ml or 54.5 μl.

 The final drug is poured 1 ml into 5 ml vials, the operations are carried out under sterile conditions in a laminar box.

 After the spill, the vials are closed with sterile plugs and sealed with foil.

The resulting drug remains active for at least 6 months when stored at 4-8 ^o C.

Claims (2)

1. A drug for the treatment of muscular dystonia, containing botulinum toxin type A, isolated from a culture of Clostridium botulinum albumin, characterized in that the botulinum toxin is isolated from a culture of Clostridium botulinum type A strain 98 in the form of a liquid substance, dissolved mainly in 0, 87% 0.005 M sodium chloride solution with a pH of 4.9-6.9 in the following ratio, mg / ml:
Sodium chloride - 0.8-1.0
10% Albumin Solution - 0.6-0.9
Substance of botulinum toxin - 0.000001-0.000005
2. A method of obtaining a medicinal product for the treatment of muscle dystonia isolated from a culture of Clostridium botulinum type A, comprising cultivating the strain on a nutrient medium, sedimentation, extraction, purification and sterilization, characterized in that the botulinum toxin type A is obtained from a culture of Clostridium botulinum strain 98, cultivation is carried out at 29-30 ^o C for 3-6 days, and then carried out in two stages of its liquid cleaning substance, the first of which is used metallosorbent mainly based on nickel and on the second floor ne - ion exchange sorbent at pH 6.1-6.9, followed by sterilization by membrane filtration. 3. The method according to claim 2, characterized in that the culture of Clostridium botulinum type A strain 98 is grown on a nutrient medium containing tryptone, yeast extract, thioglycolate, glucose and distilled water in the following ratio of components, g / l:
Trypton - 25-33
Yeast Extract - 25-33
Thioglycolate - 0.2-0.6
Distilled Water - Up to 1 L
Glucose - 4-6
2N NaOH - Up to pH 7.1-7.5