RU2413525C2 - Method for preparing drug for prostate function recovery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to technology of biologically active agents (BAA) of animal origin. A method for preparing a BAA for prostate function recovery involves grinding bull and/or puberty bull-calf prostate tissue, homogenising the intermediate product, keeping the homogenate in 3% aqueous solution of acetic acid and zinc chloride (ZnCl₂), separating water-soluble components of animal tissue cells by centrifugation to produce a supernatant to be concentrated, purified by ultrafiltration, adding glycine to the solution, sterilising and drying an end product. The solution of acetic acid is prepared of apyrogenic water; the homogenate is kept in the solution of acetic acid and zinc chloride at temperature 3-7°C for 48 h in being agitated by 0.5 portions every 4 h. The ultrafiltration process is executed in a circulation mode through a flat-chamber membrane installation with membrane filters of characteristic pore size d=300 A at temperature T≤10°C to produce a solution to be concentrated with the use of nano-filter compositional membranes. It enables to prepare an end product concentrate containing mixed ingredients of molecular weight M=5÷20.0 kDa to be dried by means of a spray-type drier.

EFFECT: method allows producing a high-yield powered therapeutically effective agent containing water-soluble ingredients of M<20,0 kDa.

4 cl, 1 ex, 2 dwg

Description

The invention relates to medicine and biotechnology, in particular the technology for the production of biologically active agents of animal origin from the prostate gland of sexually mature bulls and / or bulls and their subsequent use in medical practice, and can be used in the microbiological industry and for the treatment of patients.

A known method of obtaining a biologically active agent that restores the function of the prostate gland, which includes the isolation of water-soluble peptides by aqueous extraction of slaughtered animals from the prostate tissue, centrifugation of the extract, the isolation of the raw material from the supernatant, in which the raw material is precipitated with acetone, followed by cleaning and freeze drying of the target product see, for example, Havinson V.Kh. and others. A method of obtaining a substance that restores the function of the prostate gland. - In the patent of Russia No. 1417244, A61K 35/52, 06/30/1994 [3].

The known method does not provide a stable means.

In addition, in a known manner, an agent is obtained containing non-specific, high molecular weight peptides with a molecular weight of M 20.0 kDa, which cause non-specific reactions of the body, and the agent itself can be used only in the form of a solution. That is, a known agent obtained by a known method is suitable only for injection, and for suppositories it is unsuitable and, therefore, its therapeutic effect is also limited.

Closest to the claimed technical essence and the achieved result is a known method of obtaining a biologically active agent that restores the function of the prostate gland, which includes:

- grinding thawed raw materials using a meat grinder and obtaining a semi-finished product in the form of minced meat;

- adding to the semi-finished product a 3% aqueous solution of acetic acid and zinc chloride (ZnCl ₂ ) in a mass ratio of Q = 0.5 ÷ 0.7 g / l, in a volume ratio of n: m = 1: 5, where n is volume the amount of raw material, am is the volumetric amount of an aqueous solution of acetic acid, while

- homogenization of the semi-finished product is carried out using a rotary pulsation apparatus and receive the homogenate of the semi-finished product;

- secrete water-soluble components of the cells (CC) of animal tissue, in particular peptides;

- subsequently separated by centrifugation to obtain a supernatant;

- then the supernatant is concentrated by lyophilization - freeze-drying to a temperature of T = -35 ÷ -70 ° C, at which it is kept for t = 48 ÷ 72 hours with a subsequent increase in temperature to T = 29 ÷ 38 ° C and obtaining a concentrate;

- the resulting concentrate is dissolved in water at the rate of Q = 0.8 ÷ 1.5 l of water per 50 g of concentrate;

- clean the solution by ultrafiltration;

- and sterilize it by filtration through a membrane filter with a characteristic pore size d = 0.22 μm to obtain an ultrafiltrate in which the content of the active fraction of the peptides is Q = 4.0 ÷ 6.0 mg / ml, see, for example, Kurishchuk K .AT. and others. A method of obtaining a means of restoring the function of the prostate gland - in the patent of Ukraine No. 39022 A, A61K 35/48, 05/15/2001 - PROTOTYPE [4].

The known method provides a more stable and therefore relatively more effective therapeutic agent. However, the known method is characterized by all of the above disadvantages, in particular, it provides the means:

- which contains non-specific high molecular weight peptides with a molecular mass of M 20.0 kDa;

- the obtained biologically active agent can be used only in the form of a solution, that is, it is suitable only for injection, and is unsuitable for suppositories and due to this its therapeutic effect is also limited.

In addition, the known method is relatively ineffective, because with the same energy costs, labor costs and raw material costs in terms of dry matter, it provides a relatively insufficient yield - not more than 12% in the composition of UAS.

The problem solved by the invention is the elimination of the above disadvantages and the improvement of the known biologically active agents that restore the function of the prostate gland due to the improvement of the known method for producing this agent. This improvement of the known method simultaneously applies to:

1) the new proposed procedure for performing known operations of the method;

2) a new proposed implementation of such operations as the selection of a biologically active principle from a semi-finished product and spray drying of the obtained target product. That is, it concerns the operation of isolating a biologically active agent from the semi-finished product by standardizing the hydrolysis of the semi-finished product, that is, carrying out the hydrolysis reaction under standardized conditions, to obtain a standardized result, as well as performing the drying operation by using a spray drying apparatus.

This ensures the receipt of the specified biologically active agent with significantly improved characteristics due to the fact that:

- in the composition of such an agent, in addition to peptides, there are also other components of animal tissue cells that provide the above therapeutic effect, in particular peptides, lipids, carbohydrates, with a molecular mass of M≤20.0 kDa, which significantly increases therapeutic efficacy;

- the obtained components of the cells of animal tissue are low molecular weight and do not cause nonspecific reactions of the body, which also significantly increases therapeutic efficacy;

- finally, these components are obtained in the claimed method in a much larger amount, absolute and / or relative, than is the case in known technical solutions, in particular in the prototype, it also significantly increases therapeutic efficacy.

The task of the invention is solved by the fact that in the known method for producing a biologically active agent (ALS) that restores the function of the prostate gland, which has a specific organotropic activity - a prostatoprotective effect on the prostate gland, an indirect bacteriostatic effect on the microflora of gland secretion, the ability to normalize spermatogenesis, and the ability to simulate the state of T- and B-systems of immunity and increase the nonspecific resistance of the body, based on the stomach raw materials, and includes grinding thawed raw materials - prostate tissue of slaughtered animals - bulls and / or calves that have reached puberty, using meat grinders and obtaining a semi-finished product in the form of minced meat, adding 3% aqueous solution of acetic acid and zinc chloride to the semi-finished product (ZnCl ₂ ) in a mass amount of Q = 0.5 ÷ 0.7 g / l in a volume ratio of n: m = 1: 5, where n is the volumetric amount of raw materials, and m is the volumetric amount of an aqueous solution of acetic acid, with homogenization prefabricated lead using rotor pool lysation apparatus and obtaining a semi-finished product homogenate, the subsequent isolation of water-soluble modified components of the animal tissue cells (CC) with subsequent separation is carried out by centrifugation to obtain a supernatant, concentrating the supernatant, purifying it by ultrafiltration, sterilization by sterilizing filtration, drying the obtained target product, packing the target product into a low-cost container according to the invention

- prepare a 3% aqueous solution of acetic acid, which is added to the semi-finished product using pyrogen-free water,

- secrete water-soluble modified cell components (CC) of animal tissue by hydrolysis of the homogenate,

- centrifugation is carried out to obtain the supernatant of the hydrolyzate,

- purification is carried out by ultrafiltration before concentrating the supernatant,

- the concentration of the target product is carried out after ultrafiltration purification with obtaining the active principle,

- sterilization (sterilizing filtration) of the diluted solution of the final concentrate through a membrane filter with a characteristic pore size d = 0.22 μm is carried out to obtain the target product before drying.

The task set in the invention is also solved by the fact that the hydrolysis of the homogenate is carried out in a reactor in which the homogenate is placed and in which the homogenate is kept at a temperature T = 3 ÷ 7 ° C for a time t = 48 hours with stirring for a time t = 0.5 hour every 4 hours to obtain a hydrolyzate of animal tissue.

The problem posed by the invention is also solved by the fact that the cleaning is carried out by ultrafiltration in a circulation mode through a flat-chamber membrane unit with membrane filters with a characteristic pore size d = 300 Å at a tangential flow velocity in the membrane unit V≥1 m / s, temperature T≤ 10 ° C and pressure P = 0.2 ÷ 0.3 MPa to obtain a solution of the target product in the form of a mixture of amino acids, peptides, including glycopeptides, lipids, including phospholipids and glycolipids, and carbohydrates with a molecular weight of M = 5, 0 ÷ 20.0 kDa.

The problem is solved by the fact that the concentration of the target product is performed using nanofiltration composite membranes with a characteristic pore size d = 27 ÷ 30 Å at a tangential flow velocity V≥1 m / s, working pressure P = 0.6 ÷ 1.0 MPa and temperature T≤10 ° C to obtain the concentrate of the target product.

In addition, the task set in the invention is solved by the fact that glycine is added to the obtained final concentrate of the target product in a mass ratio q: p = 1: 2, where q is the mass amount of the concentrate in terms of dry matter, and p is the mass amount of glycine and get a solution of the final concentrate, which is filtered, then sterilized by filtration through a membrane filter with a characteristic pore size d = 0.22 μm to obtain the target product.

Finally, the problem set in the invention is solved by drying the obtained target product using a spray drying apparatus at a temperature of T = 70 ÷ 180 ° C and a speed of V = 4 ÷ 6 l / h, to obtain the target product - a therapeutic substance, i.e. current beginning.

And the packaging of the target product in the form of powder is carried out in a low-capacity container, respectively pharmacopoeial article No. VFS 42U-201 / 203-915-98 of 12/07/1998, 100 g each, followed by its use for the preparation of suppositories.

Such an implementation of the invention provides a stable biologically active agent with significantly higher therapeutic efficacy, that is, with significantly higher therapeutic characteristics.

The fact that a 3% aqueous solution of acetic acid, which is added to the semi-finished product, is prepared using pyrogen-free water, provides an increase in the therapeutic efficacy of the obtained product, since the claimed ALS contains no harmful impurities, in particular impurities of heavy metals, the presence of which even in microscopic doses has a sharply anti-therapeutic effect.

The fact that when isolating the active principle using standardized hydrolysis significantly increases the therapeutic efficacy of the obtained biologically active agent due to the use of all modified components of animal tissue cells in the active principle, namely amino acids, peptides, lipids, carbohydrates. And, in addition, this way of isolating the components of animal tissue cells that have a therapeutic effect increases the yield. That is, the use in the claimed method of the operation of separation of these components by using the hydrolysis reaction under standardized conditions significantly increases the efficiency of the entire claimed method of obtaining the claimed BAS as a whole.

The fact that purification by ultrafiltration is carried out before concentrating the supernatant also increases the effectiveness of the claimed method.

The fact that the claimed method uses nanofiltration provides an even greater increase in the therapeutic efficacy of the obtained biologically active agent, since due to this, it contains modified components of animal tissue cells with only a fixed molecular mass M = 5.0 ÷ 20.0 kDa, while while components with a molecular mass of M> 20.0 kDa and a mass of M <5.0 kDa are practically absent. That is, thanks to this, the claimed ALS contains such components that, when using (using) the drug, do not cause nonspecific reactions of the patient’s body, but give a therapeutically significantly higher effect.

Finally, obtaining a biologically active agent in the form of a powder, which in

further used for suppositories, further enhances the therapeutic efficacy of the obtained biologically active agent.

The essence of the invention is further explained by examples of its specific implementation and figures, which in no way exhaust the inventive concept, but are its specific illustration, and, in particular, schematically display:

- figure 1 - production line for implementing the claimed method of obtaining the claimed biologically active funds;

figure 2 is a table of a comparative analysis of the features of the claimed method and prototype.

The technological line for implementing the claimed method for producing ALS (Fig. 1) includes a table 1 for thawing raw materials - prostate tissue of slaughtered sexually mature animals - bulls and / or bulls, scales 2, a meat grinder 3 for making minced meat, a homogenizer 4, a reactor 5 for homogenate hydrolysis , a centrifuge separator 6 for centrifuging the hydrolyzate, a flat ultrafiltration unit 7, a concentration unit 8, a receiving tank 9 for the target product, a sterilizing filtration unit 10, a spray unit 11 drying container 12 for dry powder of the target product.

The following are examples of specific implementations of the claimed invention, which cannot be considered as such that limit the scope of the claims of the applicant, but should be considered as such that illustrate the invention as a whole and each aspect of the invention separately.

The production line (figure 1) works as shown below in example 1.

Example 1 implementation of the method

50 kg of raw materials thawed on table 1 and weighed on scales 2 — the prostate tissue of slaughtered sexually mature animals — bulls and / or gobies — were ground in a meat grinder 3, after which the minced meat was carefully mixed and homogenized twice in a rotor-pulsation homogenizer 4 to obtain a homogenate.

To the obtained homogenate was added 250 L of a 3% aqueous solution of acetic acid. The solution was prepared in a known manner, but using pyrogen-free water. Simultaneously, 150 g of zinc chloride (ZnCl ₂ ) was added to the homogenate. The components of the mixture were placed in a reactor 5, in which it was kept at a temperature of T = 3 ÷ 7 ° C for a time t = 48 h with stirring for a time t = 0.5 h, every 4 hours to obtain an animal tissue hydrolyzate as a result hydrolysis reactions.

After that, the obtained hydrolyzate was separated by centrifugation in a centrifuge 6 at an angular speed of N = 3000 ÷ 6000 rpm for 40 minutes to obtain 200 l of supernatant, as well as oil cake, which was re-hydrolyzed.

The supernatant obtained in an amount of 200 l was passed through an ultrafiltration flat-chamber membrane unit 7 with membrane filters with a characteristic pore size d = 300 Å at a tangential flow velocity in the membrane apparatus of V≥1 m / s, pressure P = 0.2 ÷ 0.3 MPa and temperature T≤10 ° С and after that for concentration of the target product through a membrane unit 8 with membrane filters with a characteristic pore size d = 27 ÷ 30 Å at a tangential flow velocity V≥1 m / s, working pressure P = 0.6 ÷ 1.0 MPa and temperature T≤10 ° C. As a result, we then obtained 40 l of a concentrated mixture of low molecular weight components of animal tissue cells - amino acids, peptides, lipids, carbohydrates with a molecular mass of M = 5.0 ÷ 20.0 kDa with a concentration of 18 mg / l, i.e. in an amount of 720 g in terms of on dry matter.

To the resulting concentrate was added 1440 g of glycine in a mass ratio q: p = 1: 2, where q = 720 g is the mass amount of concentrate, and p = 1440 g is the mass amount of glycine, and a diluted solution of the (final) concentrate was obtained.

After that, the resulting diluted solution of the final concentrate was subjected to sterilizing filtration through a membrane filter 10 with a characteristic pore size d = 0.22 μm to obtain the target product.

The resulting target product was then dried using a spray drying unit 11 at a temperature of T = 70 ÷ 180 ° C with a drying speed of V = 5 l / h to obtain the target product - a therapeutic substance, which is the active principle. With the calculation of irreplaceable 5% costs, which are several times smaller than the prototype, 2050 g of dry powder of the target product was obtained, which contained 684 g of the necessary therapeutic substance - a mixture of low molecular weight peptides, including amino acids, peptides , including glycopeptides, lipids, including phospholipids, and carbohydrates.

Dry powder of the target product was collected in a receiving container 12, from where it was packaged in a low-capacity container in accordance with the VFS 2U-201 / 203-915-98 pharmacopoeial article of 12/07/1998, 100 g each, with its subsequent use for preparation suppositories.

Preclinical and clinical trials of the claimed biologically active agent confirmed its significantly increased therapeutic efficacy compared to the prototype.

At the same time, the practical use of the claimed method for producing a biologically active agent confirmed its high efficiency due to a significant (6-fold) increase in yield, by obtaining a therapeutically more effective target product, as well as by reducing the number of technological operations of the method and their improvement.

Thus, the biologically active agent that restores the function of the prostate obtained by the claimed method has a specific therapeutic organotropic effect and is characterized by an increased content of specific low molecular weight cellular components of animal tissue - amino acids, peptides, lipids and carbohydrates with a molecular weight of M = 5.0 ÷ 20.0 kDa and the practical absence in its composition of high molecular weight cellular components of animal tissue with a molecular mass of M> 20.0 kDa.

Clinical trials of the claimed ALS obtained by the claimed method, confirmed its increased therapeutic efficacy when used as an anti-inflammatory, organotropic specific agent that has an immunomodulatory effect on the state of T and B lymphocytes.

Based on the foregoing, as well as a comparative analysis (together with the prototype) of the features of the claimed technical solutions shown in the table (figure 2), it can be argued that the claimed invention meets the eligibility criteria.

INFORMATION SOURCES

1. Havinson V.Kh. and others. A method of obtaining a substance that restores the function of the prostate gland. - In the patent of Russia No. 1417244, A61K 35/52, 06/30/1994 [1].

2. Kurishchuk K.V. and others. A method of obtaining funds that restores the function of the prostate gland. - In the patent of Ukraine No. 39022 A, A61K 35/48, May 15, 2001 - PROTOTYPE [2].

Claims (4)

1. A method of obtaining a biologically active agent (ALS) that restores the function of the prostate gland and has specific organotropic activity - a prostatoprotective effect on the prostate gland, mediated bacteriostatic effect on the microflora of the gland secretion, the ability to normalize spermatogenesis, and the ability to modulate the state of T and B systems immunity and increase the nonspecific resistance of the body, based on animal raw materials, including the grinding of thawed raw materials - tissue of the prostate gland of slaughtered animals - bulls and / or gobies that have reached puberty, using a meat grinder and obtaining a semi-finished product in the form of minced meat, homogenizing the semi-finished product using a rotary pulsation apparatus and obtaining a semi-finished product homogenate, keeping the homogenate in a 3% aqueous solution of acetic acid and zinc chloride (ZnCl ₂ ) in a mass quantity of Q ₁ = 0.5-0.7 g / l in a volume ratio of n: m = 1: 5, where n is the volumetric amount of raw materials, am is the volumetric amount of an aqueous solution of acetic acid, water department of the possible components of the cells (CC) of the animal tissue by centrifugation to obtain a supernatant, concentrated supernatant, purify it by ultrafiltration, add glycine to the solution, sterilize by sterilizing filtration through a membrane filter with a pore size d = 0.22 μm to obtain the target product and drying the target product, characterized in that for the preparation of a 3% aqueous solution of acetic acid using pyrogen-free water, the homogenate is kept in a solution of acetic acid and zinc chloride in a reactor at at a temperature of 3-7 ° C for 48 hours with stirring for 0.5 hours every 4 hours, the supernatant is purified by ultrafiltration in a circulation mode through a flat-chamber membrane unit with membrane filters with a characteristic pore size d = 300 A at a tangential flow rate in a membrane installation V≥1 m / s, operating pressure P = 0.2-0.3 MPa and temperature T≤10 ° C to obtain a solution that is subjected to concentration using nanofiltration composite membranes with a characteristic pore size d = 27-30 And at a tangent speed vial flow V≥1 m / s, working pressure P = 0.6-1.0 MPa and temperature T≤10 ° C to obtain the target product concentrate containing a mixture of components with a molecular mass of M = 5 ÷ 20.0 KDa, k glycine is added to the obtained concentrate and sterilizing filtration is carried out, the target product is dried using a spray drying unit, after which the target product is packaged in a low-capacity container. 2. The method according to claim 1, characterized in that glycine is added to the obtained concentrate of the target product in a mass ratio q: p = 1: 2, where q is the mass amount of the concentrate in terms of dry matter, and p is the mass amount of glycine. 3. The method according to claim 1, characterized in that the spray drying of the obtained target product is carried out using a spray drying unit at a temperature of T = 70 ÷ 180 ° C with a drying speed of V = 4-6 l / h. 4. The method according to claim 1, characterized in that the packaging of the target product in the form of a powder is carried out in a low-capacity container of 100 g, followed by use for the manufacture of suppositories.

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