RU2632709C1 - Neuroprotective agent from pig brain based on sodium monosial ganglioside - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: neuroprotective agent is a substance based on sodium monosialotetrahexosylganglioside obtained from the pig brain by extraction, precipitation of a gangliosides-containing mixture with a sodium hydroxide solution and its adsorption on a macroreticular ion exchange resin, followed by chromatographic separation on an ion-exchange column, selection of a fraction with sodium monosialotetrahexosylganglioside, determined by thin layer chromatography, dissolution in a mixture of trichloromethane, methyl alcohol and purified water, followed by purification using an ion exchange resin column, activated carbon, cation exchange resin column, filtration and drying, and having the following characteristics: sodium monosialotetrahexosylganglioside content of at least 96%, with a content of 19.0%-21.0% sialic acid residues based on dry matter, pH of the aqueous solution at a concentration of 20 mg per 1 ml - 5.0~6.5, the area of the impurity peak with a molecular weight of more than 5000 daltons is not more than 1.00% of the peak area of sodium monosialotetrahexosylganglioside, moisture content is not more than 5%.

EFFECT: improved compound properties.

2 ex

Description

The invention relates to the field of the pharmaceutical industry and relates to ganglioside neuroprotective agents from animal feed.

Gialliosides, which are glycosphingolipids containing sialic acid, are present in the cell membrane of mammals, especially in the membranes of nerve cells. Gangliosides play a significant role in the development of a nerve cell, growth, differentiation, and nerve repair.

In particular, a significant role in the treatment of diseases of the central nervous system is played by sodium monosial ganglioside (sodium monosialotetrahexosyl ganglioside, GM1, monosialotetrahexosylganglioside). When introduced into the body, GM1 can be incorporated into the structure of neuronal membranes and cause dynamic rearrangements in endogenous gangliosides. GM1 is known to help treat various disorders of the central nervous system and is a neuroprotective agent (Neuroprotective effects of monosialotetrahexosylganglioside, Ai-ping Xi. Et al., Neural Regen Res. 2015 Aug; 10 (8): 1343-1344). Monosialotetrahexosyl ganglioside has a protective effect on the secondary degenerative nerve after injuries. It also positively affects the parameters of cerebral hemodynamics and trauma leading to cerebral edema. Ganglioside relieves swelling of nerve cells by increasing the activity of cell membrane enzymes. Animal experiments have shown that ganglioside helps with behavior disorders caused by Parkinson's disease.

Typically, the source for obtaining ganglioside-based substances is animal feed, such as brain tissue of bulls or pigs.

For example, the patent RU 2483736 C2, publ. 06/13/2016, which describes a monosialoganglioside in the form of its sodium salt, which is obtained by a method comprising a) separating GM1 from fucosyl GM1 in a lipid mixture containing monosialoganglioside GM1 as the main ganglioside component using ion exchange column chromatography using an eluent containing potassium ions or cesium, (b) extraction of the solute from the eluted solution, (c) diafiltration of the aqueous solution of the extracted solute of step (b), (d) addition of the sodium salt and diafiltration of the resulting aqueous solution; (e) extracting GM1 in the form of its sodium salt.

International application WO 2014144953 discloses gangliosides and mixtures of gangliosides, for example GM1, and pharmaceutical products thereof derived from bone marrow cell lines and neuroblastoma cells.

US Pat. No. 4,849,413 A, publ. 1989-07-18, discloses ganglioside derivatives such as GM1 esters and amides derived from bovine brain tissue.

CN 104151372 A - 2014.11.19 describes a sodium monosialotetrahexylganglioside substance obtained from a frozen porcine brain, which is thawed, washed, homogenized, a liquid solution is added to concentrated hydrochloric acid, stirred and then centrifuged to obtain a separated brain protein; b) precipitated protein is added to methanol, homogenized, mixed and centrifuged to obtain an alcoholic extract; c) concentrated; d) the concentrate obtained previously is hydrolyzed, then centrifuged, and the supernatant liquid is collected to obtain a hydrolysis product; e) carry out ultrafiltration of the hydrolysis product repeatedly, separating substances with molecular weights above 1600 daltons to obtain a pre-pure GM1 product, then ultrafiltrate the pre-purified product GM1 and remove substances with molecular weights below 1500 daltons to obtain a pure GM1 solution; f) dried.

GM1 is known from the brain of a pig obtained by extraction with a mixture of water-methanol-chloroform and a two-stage chromatographic separation using a DEAE-sepharose ion-exchange medium and Sephacryl S-100 medium. The purified substance is homogeneous, has a purity of about 98.0%. The molecular weight measured by high-performance displacement chromatography is 30.0 kDa and 1546.9 Da for mass spectroscopic measurement by ionization by spraying in an electric field (Liujiao Bian, Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography, Biomedical Chromatography, Volume Medium 10, pages 1604-1611, October 2015). This solution may be indicated as the closest analogue.

The present invention is to obtain a neuroprotective agent from the porcine brain based on sodium monosial ganglioside with a high degree of purity, suitable for the production of effective injectable forms of ganglioside.

Sodium Monosialotetrahexosylganglioside (GM1)

The new neuroprotective agent is a substance based on sodium monosialotetrahexosyl ganglioside obtained from porcine brains by extraction, precipitation of a mixture containing ganglioiods with a sodium hydroxide solution and its adsorption onto a macroreticular ion-exchange resin, followed by chromatographic separation into an ion-exchange column of sodium isoxyl isomel isomethylene isomelose formation and chromatography, dissolving it in a mixture of trichloromethane, methyl alcohol and purified water, followed by purification using an ion exchange resin column, activated carbon, a cation exchange resin column, filtering and drying, and having the following characteristics:

- the content of sodium monosialotetrahexosyl ganglioside (C73H130N3NaO31 or C75H134N3NaO31) is not less than 96%, with a content of 19.0% -21.0% of sialic acid residues based on dry matter,

- pH of an aqueous solution with a concentration of 20 mg per 1 ml 5.0 ~ 6.5,

- peak area of impurities with a molecular weight of more than 5000 daltons not more than 1.00% of the peak area of sodium monosialotetrahexosylganglioside,

- humidity not more than 5%.

As macroreticular ion exchange resins, such as divinyl benzene or polystyrene based resins, which are sold under the trade names Dowex (Dow Chemical Co) and Amberlyte (Rohm and Haas), can be used. )

As a cation exchange resin, for example, Sepharose SP, Sepharose CM, Trisacryl M SP, DOWEX MAC-3, Fractogel EMD Amberlite, etc. can be used.

Preferably, GUDU resins are used as ion exchange resins, in particular a strong acid gel with a cation exchange resin 001 * 7, for example a polystyrenesulfonate cation exchange resin crosslinked with approximately divinylbenzene (http://www.sxresin.com/en/display.asp? id = 51). The resin of the strongly basic 1st anion exchange resin (gel) type 201 * 4 (http://www.sxresin.com/en/display.asp?id=90).

The resulting tool can be used to prepare a solution for injection, with virtually no pyrogenicity.

The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1

(1) Preparation of the extract

Trichloromethane, methyl alcohol and pork brain are placed in the extract container in accordance with the ratio (2.5-2.6): 5: 1, after stirring, the mixture is allowed to stand for 2 hours, until the upper layer of liquid in the container becomes transparent. The upper layer of liquid is mixed with a 1.5% solution of Sodium Hydroxide in a volume ratio of 5: 1. Cleavage (hydrolysis) is carried out by slow heating; when the liquid spills out, adjust the steam valve to stabilize the spill of the liquid. The temperature is controlled so that it does not exceed 80 ° C until the complete splitting and sampling of the upper layer of the split liquid. (Intermediate I).

(2) Absorption on a macroreticular ion-exchange resin and desorption of a sample of the upper layer of liquid extract

Upon absorption of the liquid extract, the flow rate is controlled within 5.00 l / min. After adsorption and saturation, the resin is washed with drinking water until a clear liquid flows out and the flow rate is controlled within 8.00 l / min. Dissolve and desorb with a solution of Sodium Hydroxide in methyl alcohol with a pH of 11.50-11.80; controlling the flow rate within 3.5 l / min; temperature 28-30 ° C; and the desorbed solution is collected in a proportion of more than 95%.

The stripped liquid is transferred to a concentrating container; mix and heat, controlling the temperature so as not to exceed 30 ° C; and concentrate to 1-3% of the initial volume. Settled concentrated solution for more than 8 hours for crystallization and separation under the action of centrifugal forces. The filter cake was washed with acetone and ethyl alcohol; after drying, a preformed ganglioside mixture is obtained.

An aqueous solution is prepared from a preformed ganglioside mixture; after mixing until complete dissolution, hydrochloric acid is added to a pH of 3.00-3.20. Heated to 75-85 ° C and subjected to transformation for 1.5-2 hours. After transformation, add acetone in a 10-15-fold volume; mix evenly, assert and leave. Centrifuged; washing the filter cake with acetone and obtaining a ganglioside mixture (Intermediate III).

(3) Separation of the ganglioside mixture

Intermediate III and the flowing phase (Trichloromethane, Methyl alcohol and purified water in a ratio of 55: 40: 4) are mixed, in accordance with a volume ratio of 1: 3, after sorption, the ion-exchange chromatographic column is washed with a flowing phase (Trichloromethane, Methyl alcohol and purified water in 55: 40: 4 ratio); elute and detect the collected solution in accordance with the method of thin layer chromatography; and then the solution is collected in accordance with the result of the determination, respectively. After the flowing liquid that meets the requirements is decompressed and concentrated under conditions of 0.06-0.1 MPa of a vacuum degree and a temperature not exceeding 35 ° C, adjust the pH to 7-8 with a saturated solution of Sodium Hydroxide; sediment, centrifuge and add acetone and after drying get the raw material (Intermediate IV).

(4) Department of Monosialotetrahexosyl Gangliosides Raw Material

Intermediate IV and the washing phase (Trichloromethane, Methyl alcohol and purified water in a volume ratio of 60: 40: 4) are mixed in accordance with a volume ratio of 1: 3, passed through an ion chromatographic column, washed. Collect the eluate according to the result of thin layer chromatography (TLC). From the solution with the TLC measurement of which Intermediate Product V was obtained, sampling of the collected liquid from each tank is required. After preparation, the sample is subjected to HPLC detection with a maximum single impurity of ≤0.5%; concentrate and defend samples with impurities ≤3.0%; remove the supernatant to obtain a monosialotetrahexogyl ganglionic raw material (GM-1 raw material).

(5) Cleaning, drying and packaging agent substance

Dissolve the raw material in the flowing phase (Trichloromethane, Methyl alcohol and purified water in a volume ratio of 70: 35: 4.1). The solution is passed through ion-exchange resins at a flow rate of 10 l / h. After adjusting the pH of the effluent to 4.50-5.50 with a saturated solution of sodium hydroxide, decompression, concentration and addition of acetone to obtain a precipitate; filtering and drying with air, get a purified raw product.

Activated carbon weighing 20% by weight is added to its 15% aqueous solution; and then the solution is filtered; the filtrate passes through cation exchange resins at a flow rate of 5 l / h; the effluent is collected and the pH is adjusted to 4.70-4.80 with saturated sodium hydroxide solution and then filtered. After distillation at 70-75 ° С, twice as much concentrated acetone is added to the cooled filtered solution; the temperature is reduced not lower than 0 ° C; the solution is settled and filtered; the precipitate is subjected to aeration and then vacuum drying for 24 hours at 0.08 MPa vacuum and at 49 ° C-51 ° C to obtain the substance of the agent. The dried final product is crushed to a powder for 30-40 minutes, packaged, labeled and placed in a storage place.

Example 2

The study of the characteristics of the obtained neuroprotective agent.

The study of the purity of the obtained substance

2 ml of water is added to 0.2 ml of the test solution containing the specified amount of element, then 2 ml of resorcinol hydrochloric acid solution (0.2 g of resorcinol is mixed with 10 ml of water, 90 ml of hydrochloric acid is added, then 0.25 ml 0 , 1 mol / l of copper sulfate), heated in a warm bath for 15 minutes, the color of the solution is blue-violet; 5 ml of n-amyl alcohol are recovered; the layer of n-amyl alcohol is blue.

Take about 20 mg of the product, add 1 ml of glacial acetic acid, heat in boiling water until completely dissolved, then add 1 drop of a solution of iron trichloride, and 1 ml of sulfuric acid is slowly poured into a boiling bath, which will cause the solution to separate into 2 layers, color 2 layers should be brown.

This product is a differential reaction of the sodium salt (Appendix IV C of the Chinese Pharmacopoeia (2010 version) volume II).

Acidity test

The product is diluted with water so that 1 ml of solution is 20 mg, in accordance with Appendix VIH of the Chinese Pharmacopoeia (2010 version, volume II), the pH value was 5.0 ~ 6.5.

In accordance with the UV method of spectrophotometry (Appendix IVA of the Chinese Pharmacopoeia 2010 version, volume II), the degree of absorption at a wavelength of 585 nm is determined and compared with a reference. The dry product contains 19.0% -21.0% sialic acid residues.

Clarity and color of the solution

The product is diluted with water so that 1 ml of solution is 20 mg, a colorless solution is obtained.

Appropriate substance

Water is added to the product and dissolved so that 1 mg is 5 mg as a test solution; 1 ml is precisely measured and placed in a 50 ml measuring cup, poured with water to the measuring scale, mixed, obtaining a standard solution. Take a standard solution of sialic acid, GD1a and GD3, add water so that a solution with a content of 1 ml of 100 μg is obtained as a fixed solution. Based on the quantitative chromatography method, 20 μl of the standard solution is taken, placed in a liquid chromatograph, the sensitivity is adjusted so that the peak of the main component of the chromatograph is about 20% -30% of the whole scale. Then 20 μl of a test solution, a reference solution and a fixed solution are measured, separately poured into a liquid chromatograph. If in chromatography the retention time of sialic acid, GD3 and GD1 in the test solution corresponds to the chromatographic peak, separately, it should not exceed more than 1/4 (0.5%), 1/2 (1.0%) and 1 (2.0% ) times the main peak of the standard solution, respectively, the main peak of the remaining single impurities should not exceed more than 1/4 (0.5%) times the main peak of the standard solution, and the sum of the peaks of various impurities should be no more than 2.5 (5.0%) times the main peak of the reference solution. In the chromatography of a test solution, any peak with an area of less than 0.05 of the main peak of the standard solution can be ignored.

Protein is determined by the folin reagent method (Appendix VII M of the Chinese Pharmacopoeia (2010 version), volume II). Based on the dry product, the protein content is not more than 1.0%.

High molecular weight impurities are determined by molecular exclusion chromatography (Appendix V H of the Chinese Pharmacopoeia 2010 version, volume II).

Chromatographic conditions and suitability of the testing system. Use hydrophilic modified silica gel as a filler of a chromatographic column (for example, TSK GEL 2000SWxl, 300 nm × 7.8 mm, 5 μm; or with similar efficiency); trifluoroacetic acid - acetonitrile - water (0.05: 40: 60) as the mobile phase, flow rate 0.7 ml / min; wavelength of 214 nm. The number of theoretical plates in accordance with peak A ribonuclease is not less than 5000, the degree of separation between adjacent peaks is not less than 3.0, based on the ratio of peak height and valley height.

Preparing a standard curve

Take ribonuclease A (molecular weight 13700), human insulin (molecular weight 5808), thymosin α1 (molecular weight 3108) and somatostatin (molecular weight 1638), separately add the mobile phase to dissolve and obtain a solution of 1 mg / ml as a standard molecular solution. Measure 20 μl of the above solution, individually pour into a liquid chromatograph, record the chromatogram, repeat 5 times, the relative standard deviation of the retention time was less than 2.0%. Take the retention time of each peak as an abscissa, the logarithm of molecular weight per ordinate, conduct linear regression, the correlation coefficient is less than 0.99.

According to tests, the peak area of impurities with a molecular weight of more than 5000 - not more than 1.00 wt. %

Methyl alcohol, acetone and trichloromethane are determined using the method for determination of residual solvent (Appendix VIIIP, Article I of the Chinese Pharmacopoeia (2010 version) Volume II). Meets the requirements. Humidity. Measured using the method of determining humidity (Appendix VIII M article A of the Chinese Pharmacopoeia (2010 version) volume II), the water content does not exceed 5%.

Heavy metals. Measured in accordance with Appendix VIII H of the Chinese Pharmacopoeia (2010 version), volume II, the content of heavy metals does not exceed 12%.

Pyrogens. Determined on the basis of XID Appendix of the Chinese Pharmacopoeia (2010 version) volume II; 2 ml are injected per 1 kg of the weight of a domestic cat, meets the requirements - does not contain pyrogens.

Determination of active component content using high performance liquid chromatography

Chromatographic conditions and suitability of the testing system. Amino silica gel is used as a filler; a solution of phosphoric acid (1 → 100) - acetonitrile - tetrahydrofuran (30:60:10) as a driving phase, to detect a wavelength of 205 nm. Take the required amount of the reference substance of sodium monosialotetrahexosylganglioside, sialic acid, GD1a and GD3, add water to obtain a mixed solution with a content of 1 mg 1 mg, 25 μg, 100 μg and 50 μg, respectively, this is a system suitability solution. Take 20 μl, placed in a liquid chromatograph. The number of theoretical plates is calculated as the peak of sodium monosialotetrahexosylganglioside and should not be less than 1000, the degree of separation between sodium monosialotetrahexosylganglioside and adjacent impurity peaks must meet the requirements.

The quantitative content is 96-98 wt.%.

The purity of the substance is more than 98.5%.

The neuroprotective effect of the agent according to the proposed invention was evaluated in animals with experimental cerebral ischemia - a model of ischemic stroke. It was found that the claimed agent reduced the mortality of rats from 30% (control) to 5% (p> 0.05) and, therefore, is an effective tool that can be widely used in the field of neurology.

Claims (1)

 A neuroprotective agent, characterized in that it is a substance based on sodium monosialotetrahexosyl ganglioside obtained from porcine brains by extraction, precipitation of a mixture containing ganglioiods with a sodium hydroxide solution and its adsorption on a macroreticular ion-exchange resin, followed by chromatographic separation on ion osyl isomethylene sodium, determined by thin-layer chromatography, dissolving it in a mixture of trichloromethane, methyl sp mouth and purified water, followed by purification using an ion exchange resin column, activated carbon, cation exchange resin column, filtering and drying, and having the following characteristics: sodium monosialotetrahexosylganglioside content of at least 96%, with a content of 19.0% to 21.0% of sialic acid residues in it based on dry matter, the pH of an aqueous solution with a concentration of 20 mg per 1 ml 5.0 ~ 6.5, the peak area of impurities with a molecular weight of more than 5000 daltons is not more than 1.00% of the peak area of sodium monosialotetrahexosylganglioside, lazhnost not more than 5%.