WO2010138024A2

WO2010138024A2 - Agent for protecting brain cells

Info

Publication number: WO2010138024A2
Application number: PCT/RU2010/000264
Authority: WO
Inventors: Александр Борисович ПОЛЕТАЕВ
Original assignee: Общество С Ограниченной Ответственностью "Медицинский Исследовательский Центр "Иммункулус"
Priority date: 2009-05-29
Filing date: 2010-05-25
Publication date: 2010-12-02
Also published as: WO2010138024A3; RU2405560C1

Abstract

The invention relates to an agent for protecting brain cells, which comprises a dominant protein extract from placental tissue with a protein fraction being produced by chromatographic separation, which includes 8 to 10 components having an average molecular weight of 15 to 200 kDa, wherein 70 to 80 % of the fraction consists of proteins having a molecular weight of approximately 70 kDa. The invention also relates to a method for producing said agent, which comprises extracting ground placenta using a low-alkaline buffer in the presence of inhibitors of the proteolytic cleavage of proteins, centrifuging the same, passing the centrifugation product through a chromatographic column including an anion-exchange carrier and balanced using the same buffer, washing the column to remove the material that has not bonded, eluating the proteins with a solution of a neutral salt, diluting the collected material with water until the pH reaches a low acidity value, and passing the diluted substrate sequentially through a column with an anion-exchange carrier and a column with a cation-exchange carrier balanced by a buffer solution up to a pH of approximately 6.0, the final product being obtained after sterilization.

Description

Brain Cell Remedy

Description

The invention relates to the field of pharmacology and is a means for protecting brain cells from ischemic and other injuries and a method for its preparation from placental tissue. The claimed tool can be used in medicine as a tool that normalizes the functions of the nervous system. Ischemic stroke is one of the most frequent manifestations of human vascular pathology, one of the main causes of death or persistent disability in people over 55 years of age. Moreover, the frequency of deaths reaches 20-30%, and in 30-40% of cases the outcome of ischemic stroke is a deep disability resulting from the apoptotic death of large populations of brain neurons, accompanied by pronounced disturbances in mental and / or motor functions (E.I. Gusev et al., 1998-2000).

The lack of noticeable progress in the treatment of ischemic stroke, despite the sufficient clarity of its main pathophysiological mechanisms, indicates the lack of effectiveness of the existing tools and methods for the prevention and treatment of this pathology and stimulates the experimental search for new, more effective drugs. Known methods for producing biologically active substances and medicines from animal raw materials (RF patent Ns 944191, 1994; a.s. SU Ns 1112606, 1996; a.s. SU Ns 1218521, 1994; a.s. SU Ns 1227198, 1986; RF patent Ns 1448443, 1994; RF patent Ns 1522486, 1993; RF patent Ns 2075944, 1997; RF patent Ns2161501, 2001; US patent 4341765, patent GB 1161896, 1969; patent FR 2583982, 1987). A known method of obtaining a drug with restoring activity in violation of brain function (RF patent Ns 1298979, 1993), which allows you to select biologically active peptides from the cerebral cortex of slaughtered animals that have a positive effect on neural functions. This preparation is obtained by processing the feedstock (gray matter of the cerebral hemispheres) with acetone at t = -10 ⁰ C: .. - 4 ° C with a ratio of brain tissue and acetone of 1: 5 for 18 ... 30 hours, homogenization, extraction homogenate with 2 ... 4% acetic acid solution for 48 ... 72 hours at pH = 3.5 ... 4.5 in the presence of zinc chloride at a concentration of 0.6 ... 2 g / l and with a homogenate ratio and acetic acid solution 1: (8 ... 4), centrifugation, precipitation of biologically active substances with acetone at t = -3 ° C ... -5 ° C and the ratio of supernatant to acetone 1: (7 ... 5), and ion exchange chromatography of the obtained precipitate on Biocarb cation exchange resin. As a result of ion exchange chromatography, a fraction is obtained that contains mainly alkaline polypeptides with a molecular weight of from 2000 to 6000 Da.

It should be noted that the method described in the patent of the Russian Federation N ° 1298979, does not allow to extract acidic and neutral polypeptides from the cerebral cortex, as well as to clean the resulting product from impurities, and the resulting final product has a relatively low therapeutic efficacy.

In the practice of treating ischemic stroke, Ceperalizin (EBEWE, Austria) is now widely used, which is a heterogeneous mixture of peptides and amino acids obtained from pig brain using standardized enzymatic treatment (Cerebrolysin: pharmacological effects and place in clinical practice. Sat reports of the 6th International Symposium, Moscow, 2002). Determined that This drug of biological origin has a certain level of neurotrophic activity and inhibits apoptosis of neurons subjected to various kinds of adverse effects. At the same time, the very fact of the unsolved problem of the prevention and treatment of ischemic stroke indicates the insufficient effectiveness of cerebrolysin (as well as any other nootropics and neuroprotective agents of biological origin or chemically synthesized today) and determines the need to develop and introduce new, more effective drugs.

The effectiveness of the drug “cerebralizin” is manifested when it is used in doses of 4-10 mg / kg with daily parenteral administration for 7-10 or more days after a stroke. With this use of the drug, there is a tendency to a decrease in the frequency of fatal outcomes and a slight decrease in the frequency of cases of severe disability compared with patients who received a placebo (V. I. Skvortsova - Cerebrolysin in the treatment of acute ischemic stroke. Cerebrolysin: pharmacological effects and place in clinical practice. reports of the 6th International Symposium, Moscow, 2002, 28-35).

An understanding of the biological foundations of the pathological process indicates that progress in its treatment can be achieved by finding and introducing more active neurotrophic and neuroprotective drugs. It is known that the source of a large number of trophic factors is the placenta, which provides and supports the rapid growth and accelerated development of nervous tissue and other organs and tissues of the developing fetus.

From the publication of the patent application of the Russian Federation Ns 2007 118 245, a peptide extract of the placenta is known containing 5.0 -35.0 mr / ml protein, 2.0 -5.0 mg / ml glycoproteins and 0.5-1.0 mg / ml Triton X100, and a method for its production, based on the extraction of biological material - the placenta, which is ground, is poured with phosphate buffer with the addition of newt, after which the extract is isolated by centrifugation with subsequent cleaning of impurities.

Also, from the publication of the patent application of the Russian Federation Ns 2004 130 760 A dated 04/10/2006, there is known a drug having neuroprotective and nootropic activity, containing proteins obtained by extraction of the placenta with a slightly alkaline buffer. This publication describes a method for the preparation of this agent by grinding human placenta, extraction with a weakly alkaline buffer, centrifugation, chromatography of a centrifuge with anion exchange media, equilibration with the same buffer, washing the chromatographic column from unbound material, followed by elution of the proteins with sodium chloride solution and sterilization by filtration through antimicrobial filters.

This method, which allows, in principle, in laboratory conditions to obtain the target product as such, in practice on an industrial scale did not allow to isolate the finished product of the required purity, devoid of ballast impurities of a protein nature.

The objective of the proposed technical solution is to obtain an effective tool that normalizes the functions of the nervous system, high purity acceptable on an industrial scale.

The solution to this problem is a means to protect brain cells from ischemic and other injuries, containing the dominant protein extract from placental tissue, obtaining, due to chromatographic separation of the protein fraction from 8 - 10 components with a total molecular weight of 15 to 200 kfla, with 70-80% of the fraction being protein with a molecular weight of about 70 kfla, and 20-30% of the fraction are proteins with a molecular weight of 15 to 60 kfl and 100 to 200 kfl . To obtain funds in accordance with the task, the gel filtration stage was replaced by an additional (final) stage of combined ion exchange chromatography. Namely, the primary eluate containing the preparation NP-3 was diluted 3 times with distilled water to reduce the concentration of NaCI, the pH of the solution was adjusted to 6.0, after which the resulting solution was passed through two series-connected columns with anion-exchange and cation-exchange carriers. The volume of carriers was taken at the rate of 1 ml of gel per 50 mg of total protein solution. The solution passing through both ion-exchange columns without binding to the carriers contained a purified NP-3 preparation, while impurity ballast products were bound with anion-exchange or cation-exchange carriers.

Thus, the method of obtaining the agent consists in the fact that the ground placenta is extracted with a slightly alkaline buffer in the presence of protein proteolytic cleavage inhibitors, centrifuged, a centrifugate is passed through a chromatographic column with an anion exchange medium, equilibrated with the same buffer, the column is washed from an unbound material, the proteins are eluted with a solution of salt eluted with a solution , the collected material is diluted with water to adjust the pH to slightly acidic and the diluted substrate is passed successively through a column with an anion exchange carrier and a column with a cation exchange carrier, balanced buffer solutions with a low salt concentration and a pH of about 6.0, after sterilization receive the finished product. The solution passing through both ion-exchange columns without binding to the carriers contained a purified preparation (NP-3), while impurity ballast products were bound to anion-exchange or cation-exchange carriers. The finished preparation (NP-3) was a protein fraction consisting of 8-10 components with a total molecular weight of 15 to 200 kDa, with 70-80% of the fraction being a protein with a molecular weight of about 70 kfl and was able to effectively protect brain cells from ischemic and other injuries.

EXAMPLES Preparation of NP-3

The fresh preparation of the placenta is extracted with a slightly alkaline buffer (e.g., 0.01 M Tris-HCl, pH 8 in the presence of commonly used inhibitors of proteolytic cleavage of proteins), centrifuged and passed through a chromatographic column with an anion exchange medium (e.g. DEAE cellulose) equilibrated with the same buffer.

The column is washed from unbound material with a 0.05 M solution of a neutral salt, for example, a 0.05 M NaCI solution in the original buffer without proteolysis inhibitors.

The desired product fraction was eluted by passing through a column of a 0.15 M NaCI solution. Collect the protein peak.

The resulting material was diluted 3 times with distilled water, adjusted to pH 6.0 and passed through two series-connected columns with anion-exchange and cation-exchange carriers (for example, DEAE-cellulose and KM-cellulose) balanced with 0.01 M buffer solutions with pH

6.O .. The volume of carriers was taken from the calculation of 1 ml of gel per 50 mg of total protein solution. The solution passed through both ion exchange columns without binding to the media contained purified preparation NP-3, while impurity ballast products were bound to anion exchange or cation exchange carriers, which could be regenerated according to standard protocols and used repeatedly.

The collected material is sterilized (for example, by filtration through microbial filters), the protein concentration is measured (for example, according to the Lowry method), packaged and stored until use at -40 ° С.

The resulting product has the following characteristics:

A) According to electrophoresis in a 10% polyacrylamide gel (in the presence of sodium dodecyl sulfate and dithiothreitol): a protein fraction consisting of 8-10 related components with a total molecular weight of 15 to 200 kDa, with 70-80% of the fraction being a protein with a molecular mass of about 70 kDa

B) The main protein component of the fraction, which accounts for 70-80% of the total protein, was identified immunochemically (using enzyme-linked immunosorbent assay and immunoblotting) as alpha-fetoprotein.

(Alpha-fetoprotein (AFP) is described in particular in the abstract of the dissertation of S. Yu. Rodionov (Perm -2003). AFP is a glycoprotein with a molecular weight of 69,000 daltons, consisting of one polypeptide chain containing about 600 amino acids and containing about 4% carbohydrates It is part of the genetic family of albuminoids, located in humans on chromosome 4. The effectiveness of the use of AFP in the practice of treatment of the following internal diseases has been investigated:

- pneumoconiosis; - bronchial asthma;

- infiltrative pulmonary tuberculosis; - autoimmune thyroiditis;

- chronic hepatitis and cirrhosis

C) The output of the active substance (drug NP-3), isolated from placental tissues) is at least 100 mg per 1 kg of feedstock.

D) The purified, sterile filtered and packaged preparation sent for storage is a pinkish transparent solution with a total protein concentration of 1 mg / ml.

Experimental verification showed that the proposed technical solution provides maximum biological stability and therapeutic efficacy of the drug, normalizing the function of brain cells, which is confirmed by the experimental results given in the examples illustrating the invention.

In experiments on laboratory animals, the following was revealed.

Example 2. The study of the possible pyrogenicity of the drug.

The study was conducted on adult outbred male rabbits weighing 2.5-3 kg by the conventional method (GL Xl, issue 2, p. 183) with a test dose of 0.25 mg per 1 kg of animal weight in 1.0 ml of isotonic 0 , 9% sodium chloride solution for injection. It was shown that drug administration was not accompanied by a rise in body temperature, i.e. the drug does not have pyrogenic properties.

Example 3. The study of the possible toxicity of the drug A) the General toxic effect of the drug was investigated in accordance with the requirements of the "Guidelines for experimental (preclinical) study of new pharmacological substances ”(2000): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the drug. B) A study of acute toxicity was conducted on white outbred male mice weighing 20-24 g. Animals were randomly divided into groups of 5 mice. The drug was administered to animals once intramuscularly at doses of 25 mg / kg, 50 mg / kg, 100 mg / kg, and 200 mg / kg in 0.25 ml of a sterile 0.9% NaCI solution. The control group animals were injected with the same volume of 0.9% NaCl solution.

C) Studies on the study of subacute toxicity were conducted on white outbred male rats with a body weight of 150-200 g randomly divided into groups of 5 animals. The drug was administered intramuscularly daily for 90 days at doses of 1 mg / kg, 10 mg / kg, 100 mg / kg in 0.5 ml of a sterile 0.9% NaCI solution. The animals of the control group were injected in the same volume with a sterile 0.9% NaCI solution. Before administration of the drug, on the 30, 60 and 90 days after the start of drug administration, the morphological composition of peripheral blood was studied in animals. There were no statistically significant differences between the studied parameters in the control and experimental groups (p> 0.1).

D) Studies on the study of chronic toxicity were carried out for 6 months on white outbred male rats with a body weight of 150-200 g randomly divided into groups of 5 animals. Animals of the experimental groups received the drug daily intramuscularly for 6 months. in doses of 1 mg / kg, 10 mg / kg, 100 mg / kg in 0.5 ml of a sterile 0.9% NaCI solution. The animals of the control group were injected according to a similar scheme to a sterile 0.9% NaCI solution in the same volume. Before administration of the drug, on animals 90 days and 180 days after the start of drug administration, the animals were examined morphological composition of peripheral blood, erythrocyte sedimentation rate (ESR) and serum total protein content according to the Lowry method. After the experiment, a pathomorphological study of the brain and spinal cord, 5 thyroid gland, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidneys, pancreas, stomach, small intestine, colon, thymus and spleen was performed. There were no statistically significant differences between the studied parameters in the control and experimental groups (p> 0.07). Thus, it was shown that the administration of the drug in all doses used was not accompanied by toxic reactions, which indicates a large therapeutic breadth of the dosage of the drug.

fifteen

Example 4. Experimental use of the drug (for a description of the methods, see (Roletaev A.B., Araróv N.A. PHARMACOLOGUOPLIPE, 2006, 3, 73-79.

(http://www.unisa.it/download/1966 145 1805116881 δ. Roletaev.pdf) 0

1. Laboratory adult rats were trained in labyrinth behavior with positive nutritional reinforcement in the Morris water labyrinth using standard techniques.

2. Laboratory adult rats were trained in the behavior of a passive 5 avoidance of a darkened compartment with electric pain reinforcement according to standard methods.

3. Laboratory adult rats were tested for behavior in the "open field" according to standard methods.

4. Bilateral 30-minute ligation of both 0 carotid arteries was performed at the level of the upper cervical divisions according to standard methods. 5. After 3 hours after model ischemia of the brain, animals were injected intraperitoneally with NP-3 at a dose of 0.2 mg / kg body weight or cerebrolysin at a dose of 10 mg / kg (active control).

6. After 7 days, the level of general motor and orientational research activity of animals and the level of reproduction of previously developed skills in them were evaluated (comparison groups: 1. rats after acute cerebral isinemia receiving cerebrolysin; 2. rats after acute brain ischemia who received NP- 3, 3. passive control rats not subjected to ischemia)

The effect of the drug NP-3 on the survival and function of the nervous system in rats subjected to cerebral ischemia (Roletaev AB, Araróv N. A. Pharmacologoplipe, 2006, 3, 73-79. (Http://www.unisa.it/download/ 1966 145 1805116881 δ. Roletaev.pdf)

Significance levels of differences: - p <0.05, - p <0.01 It is planned to introduce the drug into the human body in the form of nasal drip infusions (1 drop into each nostril), daily, for 12-14 days, although intravenous or intralumbar administration of the drug is also possible with an appropriate calculation of dosages.

Claims

Claim

1. An agent for protecting brain cells containing a dominant protein extract from placental tissue obtained by chromatographic separation of a protein fraction of 8-10 components with a total molecular weight of 15 to 200 kDa, with 70-80% of the fraction being a protein with a molecular weight about 70 kfl

2. The tool according to claim 1, characterized in that 20-30% of the fraction falls on proteins with a molecular weight of from 15 to 60 kDa and from 100 to 200 kfl.

3. The method of obtaining a means for protecting brain cells according to claim 1, which consists in the fact that the crushed placenta is extracted with a slightly alkaline buffer in the presence of protein proteolytic cleavage inhibitors, centrifuged, the centrifugate is passed through a chromatographic column with an anion exchange medium balanced with the same buffer, and the column is washed from of unbound material, the proteins are eluted with a neutral salt solution, the collected material is diluted with water to adjust the pH to slightly acidic and a diluted substrate is passed t sequentially through a column with an anion exchange medium and a column with a cation exchange medium balanced with buffer solutions to a pH of about

6.0, after sterilization receive the finished product.