RU2710367C2 - Method for producing cytokines from umbilical blood thrombocytes as substrate for developing drugs for humans and animals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and can be used for producing cytokines from umbilical blood thrombocytes. Method involves three stages: at the first stage, umbilical blood is sampled from different donors, Stabisol solution is administered in each packet with donor blood in amount of 20 % of the blood volume of the packet, each blood pack is centrifuged in a smooth program change mode, as a result of which blood is separated into thrombocyte enriched plasma and erythrocytes. Said program consists of the following modes: acceleration to 540 RCF for 5 minutes; acceleration to 900 RCF for 2 minutes; braking to 300 RCF and rotation for 2 minutes; braking up to 100 RCF and rotation for 1.3 minutes with subsequent disconnection of the centrifuge, then removing the erythrocytes from the packs and recycling them. At the second stage, all donor OTP packets are combined and all thrombocytes are concentrated, for this purpose the produced thrombocyte enriched plasma is transferred into the appropriate number of 50 ml-type falcon test tubes, centrifugation is carried out for concentration of thrombocytes in mode of 15 minutes at rate of 4000g, as a result of centrifugation a platelet concentrate and lean plasma are obtained. Oblong plasma is taken for use as a solvent, leaving 5 ml of plasma at the bottom of the test tubes, obtained thrombocyte concentrates of different donors are combined in one test tube with volume of 50 ml and diluted with depleted plasma to volume 50 ml or the solvent used is normal saline, if further plasma proteins need to be excluded; obtained thrombocyte concentrate pool is re-centrifuged in 60-minute mode at rate of 50g for removal of erythrocyte and leukocyte residues. After centrifugation, the supernatant fluid in amount of 45–48 ml is transferred into new 50 ml test tube, and 1 ml of the solution is sent to a laboratory for platelet count on an automated haematological analyzer. At the third stage, the obtained thrombocyte concentrate with a known amount of thrombocytes per ml or mcl is subjected to a storage/activation procedure, wherein the falcon with thrombocyte concentrate is lowered into a cryogenic storage of a dry type for 15 minutes at a temperature of -196 degrees Celsius, frozen product is stored in a freezing chamber at a temperature of -80 degrees Celsius, where it is stored for up to 3 years. Further activation of the product is carried out before immediate use by rapid thawing, for this purpose the falcon is placed in a water bath with a temperature of +37 degrees Celsius until complete thawing of the product, and if necessary to obtain a product with concentration of cytokines/thrombocytes less than in a frozen product, the end product is dissolved in the corresponding number of times, wherein the initial concentration of thrombocytes is used.

EFFECT: disclosed method allows to prepare a cocktail of cytokines for its further use as a substrate for preparing medicinal preparations, the method is economical and enables to produce a product with cytokine concentration of up to 1 million/mcl.

1 cl

Description

The invention relates to the medical industry, namely to biologically active substances that catalyze regenerative processes, and methods for their production.

Cytokines are hydrophilic signaling substances whose action is mediated by specific receptors on the outside of the plasma membrane. The main cytokines that catalyze regenerative processes are: transforming growth factor β (TGF-β), platelet growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), fibroblast growth factor (FGF), epidermal growth factor, vascular endothelial growth factor (VEGF) and endothelial cell growth factor. These cytokines play an important role in the processes of cell proliferation, chemotaxis, differentiation, and angiogenesis.

Currently, the following areas of medicine are actively introducing the use of cytokines as a stimulant of reparative processes:

1) Sports medicine - the treatment of fractures, injuries, sprains, regeneration of cartilage and tendons, studies show that the rate of regeneration can be increased by 20-50% with the use of plasma enriched with platelets. And this means a reduction in rehabilitation time and a return to an active life.

2) Surgery / plastic surgery - restoration of congenital defects of bone tissue, restoration of large bone defects after an accident, accidents. At the moment, the use of cytokines in implants to replace bone tissue is very promising, which significantly accelerates the healing of implants and reduces the risk of rejection. The use of cytokines in conjunction with implants allows

achieve full recovery of large bone defects, reducing recovery time.

3) Surgery / combustiology - treatment of burns, restoration of large areas of damage, treatment of superficial and deep trophic ulcers. The use of plasma cytokines as part of skin substitutes or as part of fibrin glue for trophic ulcers increases the regeneration rate by 50% or more percent. Even in cases where trophic ulcers do not heal using classical methods of treatment, which is typical for diabetes mellitus, the use of cytokines has been proven to reduce the volume of the ulcer and in most cases completely cover them, which ultimately avoids complications and further amputation.

4) Dentistry - treatment of periodontal disease / periodontitis and bone defects. The use of plasma cytokines can improve the osseointegration of implants, contributes to the recruitment of osteoblasts, thereby stimulating the formation of healthy bone tissue.

To obtain a therapeutic concentration of cytokines from platelets, it is necessary to isolate and concentrate platelets. According to published data, the necessary amount of cytokines for a clinically significant effect can be obtained only by concentration of platelets at the level of 1 million platelets per microliter of solvent. The development of such a drug includes the following steps: 1. Isolation and purification of platelet fraction; 2. development of platelet destruction procedures for the isolation of cytokines with the determination of the cytokine profile.

Also known is the composition "Compositions composed placental stem cells and platelet rich plasma, and methods of use thereof. WO 2012092458 A2 Application Number PCT7US2011 / 06778, which describes the possibility of using plasma enriched with platelets in conjunction with stem cells for use in regenerative medicine. Invention

refers to determining the correct composition (ratio) and use of cells and PRP.

There is also a method known as “Process for the preparation of serum and platelet growth factors extract” WO 1995015763 A1 Application number PCT / CA1994 / 000601, publication date June 15, 1995, which was developed only on pigs, centrifugation modes from 1000g to 3000g are used to concentrate platelets , and for activation, the ultrasonic treatment method is used, and subsequent extraction with acetone.

The known method has the following disadvantages - this is a technology that involves its use only in a place where there is a patient and only his material. Thus, this limits its use. Ultrasound activation and isolation protocol give low efficiency and high variability of results.

Closest to the claimed invention (prototype) is "Process for the preparation of platelet growth factors extract. US 5834418 A Application number US 08 / 618,560, publication date November 10, 1998, in which:

a. use the blood of pigs. Those. xenogenic material;

b. the platelet production protocol varies from 4 to 7 centrifugation steps at a speed of 1000-5000 g:

c. platelets are washed with a special solution of Plasma-Lyte A;

d. high scatter of the final platelet concentration from 100,000 to 1,000,000,000 platelets / μl.

The disadvantage of this method is the high cost and cost of the method (a lot of expensive equipment, additional materials), low repeatability and effectiveness of the method, the impossibility of use in people.

The objective of the invention is to develop an economical way to obtain cytokines from platelets of umbilical cord blood, as a substrate for the development of drugs for humans and animals.

The problem is solved by the proposed method for obtaining cytokines from platelets of umbilical cord blood, as a substrate for the development of drugs for humans and animals, containing three stages: at the first stage, cord blood is collected from different donors, Stabizol solution is injected into each package with donated blood in a volume of 20 % of the blood volume of the packet, each of the blood packets is centrifuged in the program smooth change mode, as a result of which the blood is divided into platelet-rich plasma and red blood cells, indicated ROGRAMME consists of the following modes: acceleration to 540 RCF for 5 min; acceleration to 900 RCF in 2 min; braking up to 300 RCF and rotation for 2 min; inhibition to 100 RCF and rotation for 1.3 min followed by centrifuge shutdown, then red blood cells are removed from the packets and disposed of; at the second stage, all donor OTP packages are combined and all platelets are concentrated, for which the obtained platelet-rich plasma is transferred to the corresponding number of 50 ml Falcon type tubes, centrifuged to concentrate platelets in 15 minutes at 4000 g, as a result of centrifugation platelet concentrate and depleted plasma, depleted plasma are removed for use as a solvent, leaving 5 ml of plasma at the bottom of the tubes obtained platelets The concentrated concentrates of different donors are combined in one 50 ml tube and diluted with depleted plasma to a volume of 50 ml, or physiological saline is used as a solvent if it is subsequently required to exclude plasma proteins; the resulting platelet concentrate pool is centrifuged again in 60 minutes' mode at a speed of 50 g to remove red blood cell and white blood cell residues, after centrifugation the supernatant in a volume of 45-48 ml is transferred to a new 50 ml tube, and 1 ml of the solution is sent to a laboratory for platelet counting on automated hematological analyzer, in the third stage, the obtained platelet concentrate with a known number of platelets per ml or μl is subjected to the storage / activation procedure, while the falcon with the platelet concentrate is omitted they are kept in a dry-type cryostorage for 15 minutes at a temperature of -196 degrees Celsius, the frozen product is stored in a freezer at a temperature of -80 degrees Celsius, where it is stored for up to 3 years, and further product activation is carried out before direct use by quick defrosting, for this, the Falcon is placed in a water bath with a temperature of +37 degrees Celsius until the product is completely thawed, and if necessary, get a product with a concentration of cytokines / platelets less than in a frozen product: The final product is diluted in the appropriate number of times, while the initial platelet concentration is used.

In the inventive method, it is proposed to use donor material as a substrate for cytokines. The use of cord blood as a platelet / cytokine source significantly reduces the cost of obtaining the final product (when comparing the prices for a therapeutic dose of platelet concentrate and the cost of obtaining a cord blood sample for the needs of a PK bank). The advantages of this drug is the presence of a cocktail of natural cytokines, with a significant shelf life, ready for use "but upon request".

Currently, many methods have been developed to obtain plasma enriched in cytokines. There are methods based on the use of immune responses for the isolation and purification of platelets. Such methods have good efficiency, but as a rule, require additional equipment and reagents, in addition, there are no corresponding kits that are allowed for use on the territory of the Russian Federation. The most common platelet isolation methods are based on the difference in size and weight of cell elements and different gradient centrifugation techniques are used to separate them. Most techniques designed to obtain

platelets are designed for use in transfusiology, in this regard, the presence of other cells in the drug is not critical. In the case when platelets are necessary as a substrate of cytokines, during the preparation of which all cellular elements are destroyed, the presence of other cells (red blood cells and white blood cells) will burden the final product with unnecessary impurities (cell membranes, protein, DNA, RNA, hemoglobin, cell waste products and etc.). Therefore, standard centrifugation methods will not provide the proper level of purification of the future drug. In connection with these circumstances, it is proposed to use platelet isolation methods with significant changes compared to the known ones.

The second step in the production of cytokines was the development of a procedure for the release of cytokines from platelets. According to the available literature, there are two of the most common methods for the isolation of cytokines from platelets, chemical and physical. The chemical method is a process similar to the process taking place in the body when platelets enter the site of damage. Thrombin and Ca + are used as an activator for the release of alpha platelet granules.

Being the most physiological method, it carries some disadvantages, such as the price and the presence of high doses of Ca + in the final product, which limits its use for cultural purposes. An alternative to the chemical method is the physical one, which consists in applying sequential quick freezing and thawing procedures without the use of cryoprotectants. This method is simple, does not require equipment or additional reagents, however, different sources of literature provide different information on the number of freeze / thaw cycles without providing information on changes in the number of cytokines. The availability of such information is necessary to reduce working time and production costs, as well as to

According to the literature, excessive freezing cycles can lead to the destruction of cytokines.

The next step is the purification of the final product from fibrin-forming agents and long-term storage of the product at low temperatures.

A method for producing cytokines from umbilical cord blood platelets as a substrate for the development of drugs for humans and animals is implemented as follows.

The proposed method allows to prepare a therapeutically suitable cocktail of cytokines for its further use as a substrate for the manufacture of drugs and cosmetics.

The objective of the method is to obtain the final product with a platelet concentration of at least 1 million per microliter and a residual concentration of leukocytes and red blood cells close to 0.

Due to the fact that the volume of umbilical cord blood of one person is limited (40-80 ml), the proposed method includes pooling (combining) of samples of different donors. To increase the efficiency of erythrocyte removal, Stabizole solution is added to the blood bag in a volume of 20% of the blood volume of the bag. The method is implemented in three stages:

I. The first stage (obtaining platelet-rich plasma):

At the first stage, umbilical cord blood is sampled from different donors, Stabizol solution is injected into each packet with donor blood in a volume of 20% of the blood volume of the packet, each of the blood packets is centrifuged in the program change mode, as a result of which the blood is divided into platelet-rich plasma and red blood cells, this program consists of the following modes: acceleration to 540 RCF for 5 min; acceleration to 900 RCF in 2 min; braking up to 300 RCF and rotation for 2 min; braking up to 100 RCF and rotation for 1.3 min followed by

turning off the centrifuge, then red blood cells are removed from the packets and disposed of.

II. The second stage (Association of samples and concentration)

At the second stage, all donor OTP packages are combined and all platelets are concentrated, for which the obtained platelet-rich plasma is transferred to an appropriate number of 50 ml Falcon type tubes, centrifuged to concentrate platelets in 15 minutes at a speed of 4000 g, as a result of centrifugation platelet concentrate and depleted plasma, depleted plasma are removed for use as a solvent, leaving 5 ml of plasma at the bottom of the tubes obtained platelets the concentrated concentrates of different donors are combined in one 50 ml tube and diluted with depleted plasma to a volume of 50 ml, or physiological saline is used as a solvent if it is subsequently necessary to exclude plasma proteins; the obtained thromboconcentrate pool is re-centrifuged in 60 minutes at a speed of 50 g to remove residual red blood cells and white blood cells; after centrifugation, the supernatant in a volume of 45-48 ml is transferred to a new 50 ml tube. and 1 ml of the solution is sent to the laboratory for platelet counting on an automated hematology analyzer.

III. The third stage (activation and normalization of the product)

At the third stage, the obtained platelet concentrate with a known platelet count per ml or μl is subjected to the storage / activation procedure, while the falcon with the platelet concentrate is lowered into a dry type cryogenic storage for 1 5 minutes at a temperature of -196 degrees Celsius, the frozen product is stored in a freezer at a temperature -80 degrees Celsius, where they are stored for up to 3 years, and further product activation is carried out before

direct use by quick defrosting, for this a falcon. placed in a water bath with a temperature of +37 degrees Celsius until the product is completely thawed, and if necessary, get a product with a concentration of cytokines / platelets less than in a frozen product, the final product is diluted in the appropriate number of times, using the initial concentration of platelets.

The resulting product contains a large number of biologically active cytokines / chemokines involved in regenerative processes. The use of a cytokine substrate is possible for the development of drugs, additives for culture media in cell culture. Directly the substrate can be used in its original form for use in cosmetology and the treatment of alopecia.

The proposed method allows to prepare a therapeutically suitable cocktail of cytokines for its further use as a substrate for the manufacture of drugs and cosmetics. The method is economical and allows you to produce a product with a concentration of cytokines up to 1 million / μl. The proposed method has the following advantages over PRP and other known technologies / patents:

- the method allows to obtain a complex of cytokines from a source having a higher concentration of cytokines - umbilical cord blood,

- umbilical cord blood cytokines have a significantly higher activity from the point of view of regeneration, therefore, the obtained cytokines can be used to create stable formulations and drugs that can be stored and used without the need for preparation (use off the shelf - “directly from the shelf”):

- due to the use of another source of cytokines, the method of procurement and isolation of cytokines is radically changed and differs from similar methods, which significantly reduces the cost of the final product and increases productivity.

Claims (1)

A method for producing cytokines from umbilical cord blood platelets as a substrate for the development of drugs for humans and animals, comprising three stages: at the first stage, cord blood is collected from different donors, Stabizol solution is introduced into each donated blood packet in a volume of 20% of the blood volume of the packet each of the blood packets is centrifuged in a program smooth change mode, as a result of which the blood is divided into platelet-rich plasma and red blood cells, this program consists of the following modes: acceleration up to 540 RCF for 5 min; acceleration to 900 RCF in 2 min; braking up to 300 RCF and rotation for 2 min; inhibition to 100 RCF and rotation for 1.3 min followed by centrifuge shutdown, then red blood cells are removed from the packets and disposed of; at the second stage, all donor OTP packages are combined and all platelets are concentrated, for which the obtained platelet-rich plasma is transferred to an appropriate number of 50 ml Falcon type tubes, centrifuged to concentrate platelets in 15 minutes at 4000 g, platelet concentrate is obtained by centrifugation and depleted plasma, depleted plasma is removed for use as a solvent, leaving 5 ml of plasma at the bottom of the tubes obtained platelets e concentrates of different donors are combined in one 50 ml tube and diluted with depleted plasma to a volume of 50 ml, or physiological saline is used as a solvent if it is subsequently required to exclude plasma proteins; the resulting platelet concentrate pool is re-centrifuged in 60 minutes at a speed of 50g to remove red blood cells and white blood cells, after centrifugation the supernatant in a volume of 45-48 ml is transferred to a new 50 ml tube, and 1 ml of the solution is sent to a laboratory for platelet counting on an automated hematological analyzer, in the third stage, the obtained platelet concentrate with a known platelet count per ml or μl is subjected to the storage / activation procedure, while the falcon with a thromboconcentrate is omitted they are stored in a dry type cryostorage for 15 minutes at a temperature of -196 degrees Celsius, the frozen product is stored in a freezer at a temperature of -80 degrees Celsius, where it is stored for up to 3 years, and further product activation is carried out before direct use by quick defrosting, for this, the falcon is placed in a water bath with a temperature of +37 degrees Celsius until the product is completely thawed, and if necessary, get a product with a concentration of cytokines / platelets less than in a frozen product, horse ny product is diluted in an appropriate amount of time, this time with the original platelet concentration.