RU2089204C1 - Method of preparing peptides recovering the prostate gland function - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: prostate tissue is milled, extracted with acetic acid solution at the room temperature at periodic stirring for 18-24 h. Then raw is poured in tanks and subjected for a single freezing at (-5)-(-20) C followed by thawing. Tank content is filtered, cleared and subjected for ultrafiltration on diaphragms (threshold transmission is 10 kDa). The obtained ultrafiltrate is diluted with water up to the required polypeptide content and medicinal form is prepared. Yield of the end product is increased by 5-8-times from the raw unit. EFFECT: increased yield of peptides. 1 dwg

Description

 The invention relates to the production of drugs used in medical practice, and for the production of drugs from animal raw materials.

 A new approach to the problem of correcting impaired body functions is the creation of drugs based on endogenous biologically active substances. Among them, regulatory peptides inside and intercellular messengers play an important role, carrying out the transfer of information necessary for the normal functioning, development and interaction of cell populations. It was found that regulatory peptides have a pronounced tissue specificity, restoring the functions of those organs and tissues that serve as the starting material for their production and at the same time do not have molecular species specificity, due to which the preparations obtained on their basis do not carry antigenic properties and associated side effects (12).

 The results of experimental clinical studies indicate that peptide bioregulators have pronounced anti-inflammatory, immunomodulating, antimutagenic, heratoprotective and anti-carcinogenic properties, which allows them to be used for the prevention and treatment of immunodeficiency diseases, chronic infections, age-related pathologies, etc. (2).

 For example, a complex of water-soluble peptides isolated from the prostate gland of cattle (cattle) has an organotropic effect on the human prostate and allows pathogenetic therapy of diseases of the prostate gland (4, 5).

To obtain a peptide complex from the prostate gland of cattle, a method is proposed that turns off the extraction of crushed raw materials with 5% acetic acid with the addition of zinc chloride, separating the extract from the cake, precipitating the target product with five volumes of acetone at a temperature of 5 ^° C, drying the precipitate in air, and washing with ten volumes of acetone and renaturation in water, followed by clarification and preparation of the dosage form (6). However, the peptide preparation obtained in this way contained protein contamination, for the purpose of purification from which an additional step was later introduced - ultrafiltration on membranes with a transmission threshold of 15 kD (7). At the same time, the yield of the target product from a unit of raw materials decreased by 8 times. According to the combination of features, this method is the closest to the claimed one. Its main disadvantage is the low yield of the target product (1.5 g per 1 kg of raw materials). In addition, the prototype method has significant drawbacks associated with the use of large volumes of acetone, which makes the production explosive, low-tech, requires cumbersome equipment (settling tanks, distillation columns, etc.) and huge production areas.

 The present invention solved the problem of creating a simple, technologically advanced and efficient method for producing biologically active peptides with a higher yield while maintaining the required activity and a given degree of purity.

 The peptide complex obtained according to the developed method does not contain high molecular weight protein contamination (HPLC data in Fig. 1; as well as a negative reaction to a protein with trichloroacetic acid), meets the criteria of authenticity (maximum absorption in the ultraviolet spectrum is 270 ± 5 nm) and specific activity [has a high ability to reactivate alkaline phosphatase inhibited by cystine] (8).

To achieve this goal, the prostate glands of cattle (cattle) are used, which are crushed, extracted in a solution of acetic acid, frozen at a temperature of minus 5 20 ^o C and thawed, after which the pre-clarified extract is subjected to ultrafiltration in a tangential flow on membranes with a transmission threshold of 10 cd.

In contrast to the prototype method, according to which the target product is isolated and concentrated by the method of acetone deposition, the claimed method introduces the stage of freezing the extracted raw materials at a temperature of minus 5 - 20 ^o C with subsequent thawing, which leads to a significant increase in the yield of biologically active polypeptides due to cryogenic violation the integrity of cell membranes, accompanied by the release of intracellular products. In addition, the process of freezing and thawing leads to cryostructuring of polydisperse particles of connective tissue and high molecular weight proteins, which greatly facilitates the subsequent separation of the extract from the cake.

 Another feature of the proposed method is the use for cleaning the target product of ultrafiltration on membranes with a transmission threshold of 10 kD. The choice of membranes with the indicated selectivity is determined by the molecular weight of biologically active peptides, which is 1 10 kD (2, 9).

 The above set of distinguishing features of the proposed method is not described in the literature, and their influence on the achievement of a technical result does not follow from a known level of knowledge in the field of pharmaceutical production technology.

 Example 1. 50 kg of a frozen bovine prostate is ground with an electric meat grinder, 75 l of 0.1 N acetic acid solution is added to the minced meat and extracted in the reactor at room temperature for 18 hours with constant stirring.

At the end of the extraction, the substrate is poured in 10 12 L into containers of polyethylene and subjected to a freezing procedure at a temperature of minus 5 ^o C with subsequent thawing. To accelerate the defrosting process, the tanks can be placed in a bath with hot water.

 After complete thawing, the contents of the tanks are filtered through a nylon mesh, clarified on an ASG-3M separator (6-8 thousand rpm) and subjected to tangential flow ultrafiltration on membranes with a transmission threshold of 10 kD (Pellicon unit with a PTGC membrane or Sartocon-2 apparatus with Polysulfone 10000 D filters) at a working pressure of 0.05 0.1 MPa. From 70 L of the extract, 65 L of ultraviolet light is obtained, which is diluted with distilled water so that the content of the polypeptides is 4-6 mg / ml, aminoacetic acid is added at a rate of 10 g / L, sterilized, and 1 ml of lyophilized is poured into ampoules.

 The yield of the target product is 8 g per 1 kg of feedstock.

 Example 2. 20 kg of frozen bovine prostate is crushed in a top and pour 30 l of 0.1 n acetic acid.

 The extraction is carried out in a reactor at room temperature for 24 hours with periodic stirring.

At the end of the extraction, the substrate is frozen at a temperature of minus 20 ^o C, and then thawed and filtered sequentially through a nylon mesh and filter board.

 The clarified extract is subjected to a tangential flow ultrafiltration on membranes with a selectivity of 10 kD (apparatus "PRA-4" with membranes "PA-10") at a working pressure of 0.05 0.1 MPa. Further, as in example 1.

 The yield of the target product is 8 g per 1 kg of feedstock.

Note to table:
* Reaction with trichloroacetic acid (table).

 ** The biological activity of the peptides was determined in accordance with the requirements of the regulatory technical documentation (8) by their ability to restore (c) the activity of alkaline phosphatase inhibited by cysteine. According to these requirements, the percentage of activity recovery should be at least 20% (table).

The drawing shows the HPLC of samples of biologically active peptides from the prostate gland of cattle (isocritical elution on a column with "Superosa-12"", 10x30), where
A complex of biologically active peptides obtained according to the claimed method;
B drug "Prostatilen" manufactured by JSC "Samson", St. Petersburg, obtained according to the prototype method;
At taps: 1 ribonuclease (MM 14000 D), 2 cyancobalamin (MM 1357 D), 3 merthiolate (MM 405 D).

 A comparative analysis of the products obtained according to the claimed method and the prototype method (drawing) shows the practical identity of the polypeptide fractions (peaks 1, 2) with a slightly lower content of low molecular weight contaminating fractions (peaks 3 to 5) in our product, represented by free amino acids and inorganic compounds.

 Thus, the claimed method allows to increase the yield of biologically active peptides from a unit of raw materials by a factor of 5–6, and also to exclude acetone from the technology for their production and thereby increase safety and environmental friendliness.

Information sources:
1. Ashmarin I.P. "Issues. Medical Chemistry", 1984, 30, 3: 2 7.

 2. Yakovlev G.M. et al. "Mechanisms of bioregulation", St. Petersburg, "Science", 1992, 40 pp.

 3. Yakovlev G.M. et al. "Wedge. Medicine", 1991, 69.5: 19-23.

 4. Tkachuk V.N. Gorbachev A.G., Havinson V.Kh., "Urol. And Nephrol.", 1991, 5: 40 43.

 5. Vozianov A.V et al. "Urol. And Nephrol.", 1991, 5: 43 46.

 6. RF patent N 14172441, cl. A 61 K 35/52, 1988.

 7. Technological regulations for the production of prostatylene, Leningrad, 1989, 93 S.

 8. VFS 42-2077-91, 1991, 8 pp.

 9. Morozov V. G. Havinson V.X. "Usp. Modern biol.", 1983, 96, 3 (6): 339 - 352.

Claims (1)

A method of producing peptides that restore the function of the prostate by grinding the prostate gland of cattle, treating the homogenate with acetic acid, followed by separation of the target product, clarifying it and ultrafiltration, characterized in that after treatment with acetic acid the homogenate is frozen at a temperature of minus 5 - 20 ^o С, then the product is thawed and the target product is separated by filtration, and ultrafiltration is carried out on membranes with a transmission threshold of 10 kD.

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