RU2441036C1 - Method of producing activated polyethylene oxide - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves oxidation of polethylene oxide with 2% potassium permanganate solution in amount ranging from 1.6 to 20% of the volume of the polyethylene oxide solution at temperature 80-100°C in the presence of an organic or inorganic acid, followed by removal of products of unreacted oxidising agent.

EFFECT: simple method of obtaining activated polyethylene oxide and high quality of the end product.

3 cl, 1 tbl, 4 ex

Description

The invention relates to medicine and veterinary medicine, in particular to a method for producing activated polyethylene oxide, which is used as a carrier and modifier in various methods of pegylation of natural and synthetic biologically active substances and pharmacological substances.

One of the ways to increase the efficiency of drugs with a protein structure is by chemical modification of their molecule, which consists not in actually changing their structure, but in the physicochemical transformation achieved by combining the native molecule with polyethylene glycol (PEG, synonym for polyethylene oxide). This process of combining a native drug molecule with PEG is called "pegylation". A similar chemical modification of the pharmacological preparations of the peptide structure is specifically aimed at improving their tolerance, increasing bioavailability, reducing immunogenicity, increasing their half-life and stability.

PEG as a potential object-modifier of peptide structure substances attracted the attention of researchers in the early 1970s. PEG is currently approved by the US Food and Drug Administration (FDA) as a substance approved for use in medicine (drug manufacturing), food, and cosmetology. PEG molecules are water-soluble polymers of ethylene oxide with two terminal hydroxyl groups; they (molecules) can have different molecular weights and stereochemical structures. The molecular weight of PEG molecules can vary between 300-40000 Daltons, and PEG macromolecules arranged in a chain can form both branched and linear stereochemistry. Currently, there is a fairly large number of drugs, which are pegylated biologically active peptides. In particular, many drugs were modified by pegylation, for example: L - asparaginase, adeno-zindeaminase, superoxide dismutase, interferon-alpha 2b, sTNF-RI, liposomal doxorubicin, uricase, interleukin-2, catalase, bilirubin oxidase, streptokin ( plasminogen activator), granulocyte / megakaryocytic growth factor (1-9). To modify the biologically active substance, polyethylene oxide must be activated by introducing highly reactive groups into its composition. Due to the fact that two functional groups are present in the structure of polyethylene oxides - hydroxyl and ether, the activation of polyethylene oxide can be carried out only by functionalization of hydroxyl groups, since ether bonds in polyethylene oxides are chemically highly stable. To activate polyethylene oxides, standard methods are used that are used to activate hydroxyl-containing sorbents (e.g., Sepharose). In particular, bromine cyanide, carbodiimides, N-oxysuccinimides, bis-oxirans, divinyl sulfone, trichlorotriazine, carbonyldiimidazole, benzoquinone can be used for these purposes (10). All of these reagents are highly toxic, and the purification of activated polyethylene oxide is a laborious and expensive process, since there should be no impurities of activating reagents in the final product due to the fact that the drugs are pegylated.

The known radiation-chemical method of activation of polyethylene oxide, which was used to immobilize the proteolytic enzymes Bacillus subtilis (11). The essence of the method consists in irradiating aqueous solutions of polyethylene oxide containing dissolved proteases with ionizing radiation (a stream of accelerated electrons or gamma radiation) in a dose of 1.0-2.5 Mrad. As a result of free radical reactions, polyethylene oxide binds to proteases due to carbonyl groups that are formed upon irradiation of the polymer.

In (12), the mechanism of radiation-chemical immobilization of proteases on polyethylene oxide is described and the yield of reactive carbonyl groups depending on the dose of ionizing radiation is shown.

The closest to the claimed method of the prototype is a method of producing insulin for oral administration, which consists in the following. Prepare a 1-50% aqueous solution of polyethylene oxide with a molecular weight of from 0.4 to 40 kDa. Then the solution is irradiated with high-energy ionizing radiation, mainly gamma radiation or a stream of accelerated electrons in doses that ensure the flow of free radical reactions, mainly 1.0-5.0 Mrad. Next, insulin is injected into a solution of radiation-activated polyethylene oxide to a final concentration (for protein) of 1-10 mg / ml (or for insulin activity of 10-100 IU / ml, respectively), in a ratio of polyethylene oxide: insulin equal to (1-500): 1, the mixture is stirred for 10-30 minutes to obtain a homogeneous transparent or slightly opalescent solution (13). The known method improves the therapeutic efficacy of insulin.

The disadvantages of the prototype method are:

1. When irradiating aqueous solutions of organic compounds, in particular aqueous solutions of polyethylene oxide, dextran and other water-soluble polymers, radiation-chemical reactions result in the formation of hydrogen peroxide and, accordingly, peroxide groups in the irradiated polymer, which, on the one hand, have cytotoxic effects, on the other hand, these compounds are the basis for the post-radiation reactions described in the literature, which in turn significantly reduce the stability of activir polymer, including the stability of the conjugate of the polymer with a biologically active substance. The yield of hydrogen peroxide is proportional to the dose of ionizing radiation (14). In this regard, the prototype method does not allow to achieve the stability of the activated polymer or its conjugates with biologically active substances without removing hydrogen peroxide or organic peroxides. This problem can be solved by various chemical methods, for example, ultrafiltration, chromatography, but in relation to radiation-activated polyethylene oxide in the modern scientific and technical literature there is no information about the developed methods for such purification, moreover, all of the above theoretically acceptable methods are laborious and expensive.

2. The prototype method uses radiation facilities such as gamma sources or electron accelerators. These are very complex and extremely expensive plants that require special working conditions and safety precautions, which greatly complicates and increases the cost of activating polyethylene oxide in comparison with chemical methods, even if toxic reagents are used and there is a need for complex methods of purification of the final product.

Thus, the disadvantages of the prototype method are reduced to complexity, economic inefficiency due to expensive equipment and insufficiently high quality of radiation-activated polyethylene oxide, due to the accumulation of peroxide products of radiolysis.

An object of the invention is to simplify the known method and improve the quality of the target product.

The technical task is achieved by the claimed method, which consists in the following

An aqueous 5-50% solution of polyethylene oxide with a molecular weight of 0.4-40 kDa is heated to a temperature of 80-100 ° C and oxidized with potassium permanganate in an acidic environment. For this, organic or inorganic acid is added to the heated solution of polyethylene oxide, then a 2% (by weight) potassium permanganate solution in an amount of 1.6 to 20% of the volume of the polyethylene oxide solution, the mixture is heated until manganese dioxide precipitates. The supernatant is filtered off from manganese dioxide and a solution of activated polyethylene oxide containing carbonyl groups is obtained. The yield of the target product by weight is 92% - 98% of theoretically expected.

The content of carbonyl groups in the activated polyethylene oxide solution is determined by the modified Siggia and Hann method (15) as follows: 1 ml of a saturated solution of 2,4-dinitrophenylhydrazine in a 95% ethanol solution and 0.02 ml of concentrated hydrochloric acid are added to 1 ml of the studied sample of the activated polyethylene oxide solution acids. The tubes with the reaction mixture are shaken and kept in a boiling water bath for 5 minutes. At the end of the holding time, the contents of the tubes are cooled to room temperature. Then, 4 ml of a 0.1 M aqueous solution of potassium hydroxide are added to the reaction mixture and stirred. After 10 minutes, the optical densities of the solutions were determined at a wavelength of 480 nm. As a comparison solution, a control sample prepared simultaneously with the experimental one is used, in which, instead of the activated polyethylene oxide solution, an inactive concentration solution of inactive polyethylene oxide is used. The concentration of carbonyl groups in μM / ml is determined by the calibration curve, for the construction of which acetone is used.

Table 1 presents the dependence of the concentration of carbonyl groups in activated by the claimed method polyethylene oxide 1500 on the amount of potassium permanganate solution. As can be seen from the presented results, when using a 2% potassium permanganate solution in an amount of less than 1.6% of the volume of the polyethylene oxide solution, the concentration of carbonyl groups in the polymer is significantly reduced, and when used in an amount of more than 20%, it does not increase. Thus, the optimal range of a 2% potassium permanganate solution for the oxidation of polyethylene oxide is from 1.6 to 20% of the volume of the polyethylene oxide solution.

Table 1 The amount of a 2% solution of potassium permanganate,% of the volume of a 10% aqueous solution of polyethylene oxide 1500 The content of carbonyl groups in activated polyethylene oxide, μm / mg 0.8 0.210 ± 0.018 1,6 0.620 ± 0.057 four 0.708 ± 0.086 10 0.735 ± 0.091 fifteen 0.869 ± 0.089 twenty 0.969 ± 0.103 thirty 0.931 ± 0.096

As an organic acid, for example, acetic, formic acid can be used, as inorganic acid - phosphoric acid.

When using the initial 5-50% solution of polyethylene oxide to achieve the claimed technical effect, organic or inorganic acid is added to it in a specified sequence at a concentration of 5-35% and in an amount of 0.5-2.0% of the volume of the polyethylene oxide solution; then a 2% solution of potassium permanganate in an amount of from 1.6% to 20% of the volume of the initial solution of polyethylene oxide.

The determining significant difference of the proposed method from the prototype method is that the polyethylene oxide oxidation reaction is carried out with a 2% potassium permanganate solution in an amount of 1.6 to 20% of the volume of the polyethylene oxide solution at a temperature of 80-100 ° C in the presence of an organic or inorganic acid, which allows you to significantly simplify and reduce the cost of the method, by eliminating complex and expensive equipment, and at the same time improve the quality of the target product, since in a solution activated by the claimed method PEO no peroxide compounds. The absence of peroxide compounds was determined by the Wagner method (15).

The invention is illustrated by the following examples of specific performance.

Example 1

A 50% aqueous solution of polyethylene oxide with a molecular weight of 0.4 kDa is heated in a boiling water bath to a temperature of 100 ° C, a 35% solution of acetic acid is added to it in an amount of 0.5% of the volume of the solution, the mixture is stirred and a 2% solution is added. potassium permanganate in an amount of 1.6% of the original volume. The reaction mixture is heated until a brownish-black precipitate of manganese dioxide precipitates, the supernatant is drained, cooled to room temperature and filtered, for example, with filter paper. The yield of the target product is 92%. Activated polyethylene oxide does not contain impurities of any chemical ingredients, in particular hydrogen peroxide and organic peroxides, which would have a cytotoxic effect or reduce the stability of the activated polymer and its conjugates with biologically active substances.

Example 2

A 5% aqueous solution of polyethylene oxide with a molecular weight of 40 kDa is heated in a boiling water bath to a temperature of 80 ° C, a 5% solution of phosphoric acid is added to it in an amount of 2.0% of the volume of the initial solution of polyethylene oxide, the mixture is stirred and 2% is added - potassium permanganate solution in an amount of 4% of the volume of the initial solution of polyethylene oxide. The reaction mixture is heated to a brownish-black precipitate of manganese dioxide, cooled to room temperature, filtered, for example, through a paper filter. The yield of the target product is 98%. Activated polyethylene oxide does not contain impurities of any chemical ingredients, in particular hydrogen peroxide and organic peroxides, which would have a cytotoxic effect or reduce the stability of the activated polymer and its conjugates with biologically active substances.

Example 3

A 10% aqueous solution of polyethylene oxide with a molecular weight of 1.5 kDa is heated in a boiling water bath to a temperature of 90 ° C, a 15% solution of formic acid is added to it in an amount of 1.0% of the volume of the initial solution of polyethylene oxide, the resulting mixture is stirred and added 2% solution of potassium permanganate in an amount of 10% of the volume of the initial solution of polyethylene oxide. The reaction mixture is heated to a brownish-black precipitate of manganese dioxide, cooled to room temperature, filtered, for example, through a paper filter. The yield of the target product is 95%. Activated polyethylene oxide does not contain impurities of any chemical ingredients, in particular hydrogen peroxide and organic peroxides, which would have a cytotoxic effect or reduce the stability of the activated polymer and its conjugates with biologically active substances.

Example 4

A 10% aqueous solution of polyethylene oxide with a molecular weight of 4.0 kDa is heated in a boiling water bath to a temperature of 100 ° C, a 5% solution of acetic acid is added to it in an amount of 2% of the initial volume of the solution of polyethylene oxide, the mixture is stirred and 2% is added - potassium permanganate solution in an amount of 20% of the initial volume of polyethylene oxide. The reaction mixture is heated to a brownish-black precipitate of manganese dioxide, cooled to room temperature, filtered. The yield of the target product is 94%. Activated polyethylene oxide does not contain impurities of any chemical ingredients, in particular hydrogen peroxide and organic peroxides, which would have a cytotoxic effect or reduce the stability of the activated polymer and its conjugates with biologically active substances.

Using the proposed method for producing activated polyethylene oxide will allow:

- to simplify the method through the use of affordable, cheap and non-toxic chemical raw materials (permanganates);

- eliminate the use of complex and expensive equipment;

- improve the quality of the target product by eliminating the possibility of the formation of impurities of any chemical ingredients, in particular hydrogen peroxide and organic peroxides.

Claims (3)

1. The method of producing activated polyethylene oxide, including the oxidation of polyethylene oxide and removal of the products of the reacted oxidant, characterized in that the oxidation reaction of the polyethylene oxide is carried out with a 2% potassium permanganate solution in an amount of 1.6 to 20% of the volume of the polyethylene oxide solution at a temperature of 80-100 ° C in the presence of an organic or inorganic acid. 2. The method according to claim 1, characterized in that the starting product is polyethylene oxide with a molecular weight of 0.4-40 kDa in the form of a 5-50% aqueous solution. 3. The method according to claim 1, characterized in that the acids used are organic acids, mainly acetic or formic, or inorganic, mainly phosphoric acid, in a concentration of 5-35% and in an amount of 0.5-2.0% of the original volume of polyethylene oxide solution.