WO2006014121A1

WO2006014121A1 - Muscular dystonia treatment preparation produced from clostridium botulinum culture toxin and method for the production thereof

Info

Publication number: WO2006014121A1
Application number: PCT/RU2004/000256
Authority: WO
Inventors: Jury Viktorovich Vertiev
Original assignee: Obschestvo S Ogranichennoi Otvetstvennostyu 'toksiny I Soputstvuyuschie Produkty'
Priority date: 2004-07-01
Filing date: 2004-07-06
Publication date: 2006-02-09
Also published as: RU2255761C1

Abstract

The invention relates to a preparation which is used for treating human muscular dystonias such as strabismus and torticolis, involuntary nictation and is obtainable from a Clostridium botulism culture toxin and to a method for production thereof. The inventive pharmaceutical for treating muscular dystonias comprises a botulinum toxin extracted from a clostridium botulinum culture in the form of a liquid substance dissolved mainly in a 0.87 % physiologic saline which is used for treating muscular dystonias and extracted from a clostridium culture. The inventive method consists in obtaining the botulinum toxin from the Clostridium botulinum culture by cultivating the strain thereof at a temperature of 29-33 °C for 3-6 days on a nutrition medium, in slowly adding a dry ammonium sulphate into a cultural liquid in such a way that a sedimentation is formed, in centrifuging the thus produced suspension for isolating the sedimentation and in extracting and purifying the isolated liquid phase. The purification of a liquid substance of the pharmaceutical is carried out in two stages, wherein the first stage consists in using a metal sorbent and the second stage consists in using a gel-filtration with pH of 5.5-6.0. The purified substance is sterilised by means of a membrane filtration.

Description

MEDICINE FOR TREATMENT OF MUSCLE DYSTONIA

FROM TOXIN OF CULTURE CLOSTRIDPJM BOTULINUM

AND METHOD FOR ITS PRODUCTION

O L A T T E X N I K

The invention relates to a chemical - pharmaceutical industry ¬

l is concerned with the creation of a drug intended for treatment

muscular dystonia of a person, such as strabismus, torticollis, nepr¬

arbitrary blinking, etc.

BACKGROUND OF THE INVENTION

Known from Schaptz EJ., Johnsop E.A. Рrorertiеs apd usе оf botuli-

Pum Tohp Apd Other Peurotokhips IP Medisip. Misrobio.Rev., 1992, 56, 80-

99 medicinal product (the preparation "BOTOX" is used for treatment

muscle dystonia) and method for its production on the basis of botulinum

type A toxin, including the cultivation of strain Clostri-

botulinum type A, toxin sedimentation along with bacterial

cells at pH 3.7, extraction with IM NaCl, precipitation with ethanol (final

concentration (15%), dissolution in phosphate buffer and subsequent ¬

crystallization during dialysis against 0.9 M ammonium sulfate. By-

barely recrystallized botulinum toxin diluted in 0.15

M NaCl to a concentration of 100Ld ₅ (/ ml, add serum albumin

min person to a concentration of 500 μg / ml, sterilized and lyophilized

yut. 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. Known from Das Gurta BR, Sathuamoorthu V. Rshifϊsatiop apd ami- no asid somrositiop ° F ture A botulipum peurotohip. Toxisop, 1984, 32, 415–434, a drug for treating muscular dystonia, containing botulinum toxin type A, isolated from a Clostridium botulipum culture and albumin albumin and a method for producing a medicament for treating muscular dystonia, including cultivating the strain on a nutrient medium , precipitation, extraction, purification using DEAE-cellulose, sterilization and lyophilization.

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

The closest in their essence and achieved effect to The proposed medicinal product for the treatment of muscular dystonia and the method for its preparation are known from the patent of the Russian Federation JУfe 2206337, Cl. ⁷ A 61 K 39/08, 2002, a medicament for treating muscular dystonia containing a botulinum toxin isolated from a Clostridium botulium culture and albumin, and a method for producing a medicament for treating muscular dystonia isolated from a Clostridium botulir culture, comprising cultivation of the strain on a nutrient medium, sedimentation, extraction, purification and sterilization. The disadvantages of this drug for the treatment of muscular dystonia and the method of its production is that it is stored for no more than 6 months, and its receipt is accompanied by an increased specific consumption of materials.

DISCLOSURE OF THE INVENTION The objectives of the invention is to obtain a medicinal product for the treatment of muscular dystonia that does not lose its activity for at least two years when stored in liquid form and to reduce the specific consumption of materials necessary for its production. To solve this problem, a group of inventions is proposed, united by a common inventive concept. These objectives are achieved in that the drug for the treatment of muscular dystonia, containing botulinum toxin isolated from a culture of Clostridium botulirшm and albumin, consists of botulinum toxin type A strain 98, or type B strain N ° 364, type C strain N ° 20, or type E strain N2I88, or type F strain N_> 470, or type G strain N_> 89, or mixtures thereof in various combinations in the form of a liquid substance dissolved in not more than 0.87% saline solution with a pH of 4.9-6, 9 with the following ratio of components, mg / ml: Sodium chloride - 0.8 - 1.0

10% albumin solution - 0.6 - 0.9 substance of botulinum toxin - 0.000001 - 000005, and in the method for producing a medicinal product for the treatment of muscular dystonia from the toxin of the Clostridi culture botulipum culture, including cultivating the strain on a nutrient medium, sedimentation, extraction purification and sterilization by membrane filtration, botulinum toxin type A, or type B, or type C, or type E, or F, or G are obtained from Clostridium botulipum culture, respectively, from strains ЖN_> 98, 364, 20, 188, 470 and 89, which culturing is carried out at a temperature of 29 - 33 ⁰ C in those ix 3 - 6 days, and then carried out in two stages of purification of liquid substances, the first of which The use metallosorbent, and in the second stage - gel filtration at pH 5.5-

6.0, after which sterilization by filtration is carried out

through a membrane, the pore sizes of which are 15 - 25 microns, obtained

various botulinum toxins, if necessary, are mixed in various

ny combinations ..

In addition, in a method for producing a medicament for

for treating muscular dystonia from the toxin of the Clostridium botulipum culture

as a metal sorbent in the first stage of purification can be used

nickel-based metal sorbent was used, and Clostridium botu- culture

lipum is grown on a nutrient medium, the following composition:

Trypton - 25 - 33 g / l

Yeast extract - 25 - 33 g / l

Thioglycolate - 0.2 - 0.6 g / l

2 n NaOH - up to pH 7.1 - 7.5 g / l

Glucose - 4 - 6 mg

Distilled water - up to 1 liter.

BEST OPTION FOR IMPLEMENTATION FROM OVERGROUND Frozen are used for the preparation of seed

uterine cultures of ZhNs strains 98, 364, 20, 188, 470 and 89 Cl-stridium

botulipum. Each uterine culture in the amount of 0.5 ml is sown on

tryptone-yeast medium, poured into 50 ml bottles

100 ml The cultures are grown at a temperature of 33 ⁰ C for 3 to 5 days current. Sowing materials can be used for sowing for 6 months; stored at 4 ° C. From vials with uterine cultures, crops are sown on TD medium in order to obtain a native toxin: 2.5 ml of inoculum is added to 2.5 L of sterile tryptone-yeast medium and each individual culture is grown in an incubator at a temperature of 33 ⁰ C within 6 days.

Then determine the toxicity of the obtained culture fluids. For each strain, 2 samples of its culture fluid, 1.0 ml each, were 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 within 5x10 ⁵ DLM / ml, it is diluted sequentially with a tenfold interval to 1x10 ⁵ and then 2x10 ⁵ , 4x10 ⁶ . 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. At this stage, the type specificity of each strain is also determined. The essence of the determination of type-specificity is shown in redefining it for type A toxin strain Ns98. For this, the culture fluid after trypsin activation is diluted to a concentration of 100,000 DLM / ml and the neutralization reaction is performed on white mice with diagnostic anti-botulinum sera A, B, and E. 1 ml are poured into 3 tubes prepared dilution of the culture fluid with a concentration of 100,000 DLM / ml. In the first test tube add 1 ml of diagnostic serum type A, in the second - type B, in the third - type E (all serums 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 at least 4-7x10 ⁵ DLM / ml. To the resulting volume of the culture fluid in the bottle add 3 NH ₂ SO ₄ to pH 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 to 18 hours at room temperature. temperature to form a precipitate. The culture fluid is centrifuged at 8000 rpm for 10 min at 4 ° C. The supernatant is drained and the precipitate is used for further work. To extract the toxin, use 0.2 M acetate buffer pH

6.5 - 6.7 containing 1 M NaCl. An extraction buffer is added to the precipitate (1/20 of the initial volume; for example, 125 ml of the buffer is added to the precipitate from 2.5 L of culture liquid), suspended by careful pipetting and extracted for 1 hour with constant stirring. The suspension is centrifuged for 10 min at 4 ^° C. The ha-supernatant is poured into a clean chemical beaker and the precipitate is discarded. At this stage, receive 120-135 ml of the drug with activity from 6x10 ⁶ to 1.2 x 10 ⁷ DLM / ml.

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 ^° C for 2 years. The suspension is centrifuged at 15,000 rpm for 10 min at 4 ^° C. The supernatant is drained and the precipitate is used for the latest 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 preparation is centrifuged at 15,000 rpm for 10 minutes at 4 ^° C. The supernatant is poured into a clean beaker. 10 ml of a 50% suspension of the metal of the sorbent is added to the solution of the toxin and incubated for 30 min with constant stirring in a shaker at 60 rpm and a temperature of 4 ⁰ C. Then, the metal sorbent is precipitated by centrifugation for 5 min at 3000 rpm, and the supernatant the liquid is discarded. To wash the sorbent from an unbound toxin, 10 ml of 50 mM phosphate buffer pH 6.8 is added to the sorbent and the centrifugation step is repeated. To elute the toxin, 5 ml of elution buffer (0.05 M phosphate buffer pH 6.5, 0.05 M NaCl, 0.05 M EDTA) is added to the sorbent, incubated for 15 min at 22 ⁰ C with constant stirring at 30 rpm . The suspension is then centrifuged at 3000 rpm for 5 minutes. The supernatant is 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 50 cm (column volume 50 ml) is filled with 50 ml gel filtration gel - Serhasrul S-300 and 56

10

100 ml of 0.1 M ρH-5.5 citrate buffer are collected. The toxin solution is applied to the column using a peristaltic pump at a rate of 1 ml / 1 min.

The toxin is eluted with ml of 0.1 M citrate-phosphate buffer pH 5.5. Fractions were collected in 1 ml sterile glass tubes. Fractions containing purified toxin are pooled and labeled as purified botulinum toxin type A. The purified toxin is placed in a syringe and filtered through a 0.22 μ filter nozzle into a sterile glass tube in a laminar box. At this stage, the protein is determined in the preparation, activity in

DLM in mice, activity in LD _5O and specific activity.

The protein is determined spectroscopically at 280 nm, based on the fact that the concentration of purified botulinum toxin of 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 ranges from 3xlO ⁶ to IxIO ⁷ DLM / 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 3xlO ⁶ to IxIO 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 _5O 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 are used, the body weight of which is in the range from 17 to 23 g.

To determine Ld50 use 2 ml of the drug. 1.8 ml of physiological saline are added to each 1 ml of the preparation, and then the following dilutions are made: Dilution 0: 4.4 ml of gelatin-phosphate buffer is added to 2.2 ml of the preparation;

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 was 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 should have the same number of animals, b) in the group that was injected with the most concentrated preparation, 100% animal death should be observed, in ) in the group to which the least concentrated preparation was administered, animal death should not be observed; d) all solutions should have a constant dilution ratio. LD ₅₀ is calculated by the formula: m = L - d (Σpj-0.5) where:

LD ₅₀ - breeding, in which the death of 50% of animals occurs m - logarithm of LD50 (log LDso) L - logarithm of breeding the highest concentration of the drug in the introduced series d - log dilution factor

pj - the share of animal death in j breeding

∑рj - the sum of the shares of the death of animals

The standard error is calculated using the following formula:

Standard error = - n - 1

Where:

∑ [pj (1 - pj)] is the sum of the shares of the death of animals at j breeding,

multiplied by (1 - the sum of the shares of the death of animals at j breeding)

p - the number of animals per group for the test dose

95% confidence interval is calculated according to the following form

mule:

Upper bound = M + (1, 96 x standard error)

Lower bound = M - (1.96 x standard error)

Results are converted to LD ₅₀ units using anti¬ tables

logarithms. Due to the fact that only 0.1 ml of plant is injected into each mouse

thief, LD50 units are multiplied by 10 to express the result

tats in units of LD ₅₀ per 1 ml.

The activity of the purified preparation of botulinum toxin co¬

sets from 9x10 ⁶ to 3xlO ⁷ LD ₅ () / ml.

The specific activity of the drug is calculated by the formula: The specific activity (LD ₅ o / mg) = the amount of protein mg / ml The specific activity of the purified preparations of botulinum toxin is from 3 x 10 ⁷ to 1 x 10 ⁸ LD ₅₀ / mg.

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

The dilution factor is calculated by the following formula: V _b x U _e

X = V _f ^χ U _s 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 bottle, 56

fifteen

U _e is the number of units in the bottle, U _s is the number of units / ml in V _b

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 x 10 ⁶ units / ml, then

1200 xlOO = 120000 X = l ^χ 2.2xlO ⁶ = 2.2 ^χ lO ⁵ X = 0.545 ml

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

Operations are carried out under sterile conditions in a laminar box. After filling, the bottles are closed with a sterile stopper and clogged with foil.

The resulting preparation retains its activity for at least 2 years when stored at 4-8 ^° C.

Examples of the method for producing a medicinal product for the treatment of muscular dystonia: Example 1.

The composition of the nutrient medium for the cultivation of Clostridium botulinum, strain B 364:

Trypton 30 - 33 Yeast extract 25 - 33

Sodium thioglycolate 0.5 - 0.6

2 N NaOH to pH 7.3

Glucose 5 Diet, water up to 1 liter

The medium is made from commercial ingredients that are not of animal origin.

The prepared medium is autoclaved at 110 ^° C for 30 minutes at 0.5 atm. For the preparation of inoculum, a frozen uterine culture of the Clostridium botuliшдm strain is used. A uterine culture in the amount of 0.5 ml is seeded on trypto-yeast medium, poured into 50 ml in 100 ml bottles. 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 ° C for 4-5 days. Seed can be used for sowing for 6 months; stored at 4 ^° C. From vials with a uterine culture, inoculation on TD medium is performed to obtain a native toxin.

Before use, 40% sterile glucose solution is added to the culture medium to a final concentration of 0.5%. In 2.5 l of sterile tryptone-yeast medium, 2.5 ml of seed T / RU2004 / 000256

17

and the culture is grown in a thermostat at a temperature of 33 ⁰ C for 6 days.

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 with gelatin-phosphate buffer in accordance with the intended titer. For example, to determine the toxin titer within 5><10 ⁵ DLM / ml, it is diluted sequentially with a ten-fold interval to IxIO ⁵ and then 2x 10 ⁵ , 4x10 ⁵ . From each dilution, 0.5 ml of the toxin is injected intraperitoneally into two mice weighing 16-3 Or, 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. Determination of type specificity is carried out after determination of toxicity by the corresponding NTD.

The culture fluid is used in further work if the toxin contained in it is strictly type-specific and the toxicity of 1 ml of the solution is at least 4 - 7xlO ⁵ DLM / ml for types A and B, 4 - 7x 10 ⁵ DLM / ml 1 - 2 xlO ⁵ DLM / ml for types E and F and 0.4 - O, 6xlO ⁵ DLM / ml for type G. 3 NH ₂ SO _{4 is} added to the resulting volume of culture fluid in the bottle 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 at pH

- meter. The mixture is left for 2-18 hours at room temperature to form a precipitate.

Then carry out centrifugation, while before centrifuging the precipitate, the centrifuge and the rotor are cooled to 5-7 ⁰ C. Prepare dishes for draining the centrifuge.

The culture fluid is placed in 500 ml beakers and centrifuged in a medium speed centrifuge at 8000 rpm for 10 minutes at 4 ⁰ C. The supernatant is poured into a bottle and the precipitate is used for further work. For the extraction of the toxin, 0.2M acetate buffer pH 6.5-7.0 containing IM NaCl is used. A 0.2M solution of acetic acid is prepared: glacial acetic acid 1.13 ml, distilled water - up to 100 ml. A portion of sodium acetic acid 3H ₂ O is made 27.2 g, sodium chloride - 58 g, to which 4 ml of a 0.2M 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 the buffer is added to the precipitate from 2.5 L of culture liquid), carefully maintained by pipetting and extracted for 1 hour with constant stirring.

Before centrifuging the precipitate, the centrifuge and rotor are cooled to 5 - 7 ° C. Cookware is prepared to drain the centrifuge. The suspension is placed in 250 ml centrifuge glasses and centrifuged in a medium speed centrifuge at 10,000 rpm for 10 minutes at 4 ^° C. The supernatant is poured into a clean chemical beaker and the precipitate is discarded.

At this stage, receive 120 - 125 ml of the drug with activity from 6 x 10 ⁶ to 1, 2 x 10 ⁷ DLM / ml. 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, carefully mixed with a glass rod. To form a precipitate, the solution is left for 17-24 hours at a temperature of 5-3 ° C, 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 ⁰ C for 2 years.

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

To a precipitate of 60 ml of toxin add 40 ml of starting phosphate- pH 6.8 buffer, mix thoroughly with a glass rod until the precipitate is completely dissolved and bring up to 50 ml with 50 mM phosphate buffer pH 6.8. The suspension is placed in a centrifuge and centrifuged at 15,000 rpm for 10 min at 4 ^° C. The supernatant is poured into a clean beaker.

10 ml of a 50% suspension of nickel-based sorbent are added to the toxin solution and incubated for 60 minutes at 4 ^° C and with constant stirring in a shaker at 30 rpm. Then, the metal-sorbent is precipitated by centrifugation for 5 min at 3000 rpm, and the supernatant is discarded. To wash the sorbent from an unbound toxin, 10 ml of 50 mM phosphate buffer pH 6.8 is added to the sorbent and the centrifugation step is repeated. To elute the toxin, 5 ml of elution buffer (0.05 M phosphate buffer pH 6.5, 0.05 M NaCl, 0.05 M EDTA) is added to the sorbent, incubated for 15 min at 22 ⁰ C with constant stirring at 30 rpm . The suspension is then centrifuged at 3000 rpm for 5 minutes. The supernatant is used for further work.

Low molecular weight precipitates are removed by gel filtration. A column measuring 1.0 x 50 cm (column volume 50 ml), previously washed and mounted in a tripod, is filled with 50 ml of Serhacrul S-300 gel and washed with 100 ml of 0 DM citrate-phosphate buffer pH 5.5. Operation lead at 4 C. A toxin solution is applied to the column using a peristaltic pump at a rate of 1 ml / 1 min.

The toxin is eluted with O, IM citrate-phosphate buffer pH 5.5. Fractions were collected in 1 ml sterile glass tubes. Fractions containing purified toxic complex combine the volume and are labeled as purified botulinum toxin of the appropriate type.

The purified toxin is placed in a 10 ml syringe and filtered through a 0.22 μ filter nozzle (COSTAR, SAMBRIDGE, NA, Cat. JV 8110 Corporation, NY, 1431) in a sterile glass tube in a laminar box.

At this stage, the protein is determined spectroscopically in the drug, activity in DLM in mice, activity in LD ₅ o, and specific activity. The specific activity of sensed botulinum toxin drugs is from 3 x 10 ⁷ to 8xlO ⁷ LD ₅ o / mg for types A and B, from 1 xlO ⁷ to 3xlO ⁷ LD ₅ o / mg for types C, E, F and G .

For the preparation of the final drug, use isotonic 0.9% Sodium Chloride Injection Solution manufactured by Vostok OJSC, Vostochny settlement, Kirov Region or 0.05 M citrate-phosphate buffer pH 5.5 and 10% Albumin Solution intravenously produced by the GUL 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. The dilution factor is calculated by the following formula:

V _b XU ₆

Where:

X is the number of ml of substance, Vb is the volume of the series of the drug (ml)

V _f - the volume of the drug in the bottle,

U _e is the number of units in the bottle,

U ₅ - the number of units / ml in V

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 ^χ l O ⁶ u / ml, then 120 x 100 = 120,000

X = 0, l x 2.2 ^χ lθ ⁶ = 2.2 x lО ⁵

X = 0.0545 ml or 54.5 μl, The final drug is poured 1 ml into vials with a volume of 5 ml of the operation is carried out under sterile conditions in a laminar box.

After filling, the vials are closed with sterile plugs and covered with foil.

The resulting medicinal product retains its activity for at least 2 years when stored at 4 - 8 ° C. Example 2

Composition of culture medium for cultivation of Cstridium botulipum Strains of type B Ne364 Clostridium botulipum and type E N ° 188 Clostridium botulipum were cultured on medium, and each toxin was concentrated and purified as described in Example 1.

Each purified toxin is placed in a syringe and sterilized through a 0.22 μ filter with a sterile glass tube in a laminar box. In a sterile toxin preparation, the protein content of the mouse DLM activity, LD ₅₀ activity and specific activity were determined.

The specific activity of purified botulinum toxin drugs is from 3 x 10 ⁷ to 8xlO ⁷ LD ₅ o / mg for type B, from 1 xlO ⁷ to 3xlO ⁷ LD _5O / mg for type E. For the preparation of the final drug, use "Isotonic 0.9% Sodium Chloride Injection Solution" or 0.05M Citrate-Phosphate Buffer pH 5.5 and "10% Intravenous Albumin Solution". 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. To the resulting solution, 60,000 units of purified type B toxin and 60,000 units of purified type E toxin are added.

The resulting solutions of preparations of toxins of type B and E are mixed: to 1200 ml of a solution of toxin type B with an activity of 100 Units / ml add 1200 ml of a solution of toxins of type E with an activity of 100 Units / ml. The result is 2400 ml of a drug with an activity of 100 IU / ml containing botulinum toxin type B and type E with an activity of 50 IU / ml. The final drug is poured 1 ml into vials with a volume of 5 ml of the operation is 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 medicinal product retains its activity for at least 2 years when stored at 4 - 8 ° C. INDUSTRIAL APPLICABILITY The proposed drug for the treatment of muscular dystonia from the toxin of the Clostridium botulipum culture and the method for its preparation can improve the quality of the drug and increase its activity period up to 2 years.

Claims

CLAIM

1. A drug for the treatment of muscular dystonia, containing botulinum toxin isolated from Clostridipotulipum culture and albumin, with the fact that it contains botulinum toxin type A strain 98, or type B strain G364, type C strain N ° 20, or type E strain NsI 88, or type F strain N ° 470, or type G strain N289, or mixtures thereof in various combinations in the form of a liquid substance dissolved in not more than 0.87% physiological solution with a pH of 4.9 - 6.9 in the following ratio of components, mg / ml: Sodium chloride - 0.8 - 1.0

10% albumin solution - 0.6 - 0.9 substance of botulinum toxin - 0.000001 - 000005.

2. The method of obtaining a medicinal product for the treatment of muscular dystonia from the toxin of the Clostridium botulipum culture, including cultivating the strain on a nutrient medium, sedimentation, extraction, purification and sterilization by membrane filtration, with the exception of that botulinum toxin type A, or type B, or type C, or type E, or F, or G is obtained from a Clostridipotulipum culture, respectively, from strains N ° N ° 98, 364, 20, 188, 470 and 89, cultivation which are carried out at a temperature of 29 - 33 ⁰ C for 3 to 6 days, after which they are carried out in two stages cleaning their liquid substances, in the first of which metallosorbent is used, and in the second stage, gel filtration at pH 5.5-6.0, then sterilization is carried out by filtration through a membrane, pore sizes of which are 15-25 microns, the obtained botulinum toxins if necessary, mix in various combinations ..

3. The method according to claim 2, characterized in that nickel-based metal sorbent is used as the metal sorbent in the first purification step.

4. The method according to claims 2 or 3, characterized in that the culture of Clostridi botulinum cultivated on a nutrient medium of the following composition:

Trypton - 25 - 33 g / l

Yeast extract - 25 - 33 g / l Thioglycolate - 0.2 - 0.6 g / l 2n NaOH - up to pH 7.1 -7.5 g / l

Glucose - 4 - 6 mg

Distilled water - up to 1 liter.