EA018116B1 - Process for preparing polyethyleneglycol covalent conjugate with erythropoietin - Google Patents

Abstract

The invention relates to a process for preparing polyethylene glycol covalent conjugate with erythropoietin, characterized in that: a) a free hydroxyl group of monomethoxypolyethylene glycol is acidified with halogen anhydride of nitrobenzoic acid in anhydrous pyridine; b) a nitro group of the prepared methoxypolyethylene glycol ester of the nitrobenzoic acid is reduced with chromium salt(II); c) the prepared methoxypolyethylene glycol ester of aminobenzoic acid is dinitrided with nitrite of alkaline or earth metal in a aqueous or water-organic medium or with organic nitrite in the medium of polar organic solvent with subsequent removal of nitrite excess and preparing activated pegylating agent; d) the activated pegylating agent is introduced without separation into reaction of nitro combination with erythropoietin in the aqueous or water-organic medium with pH from 7.0 to 9.5; and e) a mixture of the pegylation products is separated using ion-exchange chromatography to prepare monopegylated erythropoietin. The technical result provides broadening of erythropoietin spectrum with increased time of circulation thereof in the blood flow.

Description

The invention relates to polymer chemistry and biotechnology and provides a method for producing a covalent conjugate of polyethylene glycol with erythropoietin, in which the polymer is attached to erythropoietin via an azo group.

State of the art

Erythropoietin (EPO), a glycoprotein consisting of 165 amino acids, is physiologically the main regulator of erythropoiesis, i.e. stimulates the formation of red blood cells from late progenitor cells and increases the release of reticulocytes from the bone marrow. It promotes the proliferation and differentiation of erythroid germ cells, and also prevents their apoptosis. To perform the latter function, the concentration of erythropoietin must be maintained at a certain constant level for each person. Until tissue oxygenation is impaired, the concentration of erythropoietin, as well as the volume of circulating red blood cells, remain constant (Viešlj M., Köökötb. Netayu Adeyk Meb Syet. 2006; 4 (4): 299-311; Soobpoidy T.T., 8k1kpe V., Vgidpaga S Egu1ygoro1eyp, 1gop, apb egu1ygoroek1k // B1ob. 2000; 96: 823-833; Ca1,,. Mapa1o Ό.Ι, Ve1 S. e1 a1. Neike cat gobeck exrokeb ш 1 ег т т й ог аг аг 1 1 1 1 1 г аг г г ерей ерей ерей иг иг иг иг иг. SlgsikLyup. 2003; 108: 79-85; Ermolenko V.M., Nikolaev A.Yu. Erythropoietin: biological properties and clinical use. Ter. arch. 1990; 62 (11): 141-145; Egk1et A.1., Sago 1., Kapki E., 8begg V. Vepa1 apb ex! Gagepa1 egu111goro1ebp rgobiyyup sh apayat gak. Br 1 NayetaFk 1980; 45 (1): 65-72; E1ake A.U., Natkop M.V., Abkhuk Ν.8., Ζаη ^ аη ^ ЕУ. Hegu111goro1ebp rgobiyyup Ly 1 Ge1a1 Yueg sh ap abiY epuyoptey. B1ob. 1987; 70 (2): 542545).

Clinically, EPO is used to treat anemia in chronic renal failure (dialysis and pre-dialysis patients), in oncological diseases (cytostatic therapy), in organ and tissue transplantation, in the complex treatment of AIDS (HIV therapy with zidovudine), in auto donation, in the treatment of anemia and in chronic inflammatory diseases, in case of anemia in debilitated patients (elderly people, premature babies, burnt, etc.) and in refusal from transfusions of allogeneic hemocomponents (Wake S., Soobpoidy L.T. Apet1a o Mr. Sigoys Ekeake // No. \ ν. Unit 1. Furniture 2005. yo1 352. No. 10. R. 1011-1023; KaЙtakkeg 1.R., Kekk1eg i., Co11ksya1k V. 81isk1 S., Moyeg B. EGGes! OG gesotipai yitap eGu! YGOROEIP APB SHYAUEPOIK 1GOP OP APET1A APB BKEAKE AS! 1U11U Т ГЕЕИТА1О1Б аПЕПЬК // 1. ВееитаФк 2001; 28: 2430-2436; // Sigg.NetUyk Ver. 2003; 2: 116-121; Migryu 8.T., RagGgeu R. 8. Thie 1tray oG apet1a soggeyup op sagbyuaksyag bkeake t epbk! Ada hepa1 bieake // 8ett. No. Rygo1. 2000; 20: 350-355).

The bioavailability of commercial preparations based on recombinant erythropoietin is limited due to the short half-life in plasma and the tendency to degradation by proteases (Pavlov A.D., Morshchakova E.F. Regulation of erythropoiesis: Physiological and clinical aspects. M: Medicine, 1987. 272 p. ; Vate A. Ye. S., fuck. Abuapseb hepa1 tebut. OhGogb: OhEogb iyuegkyu rgekk, 1992. 484 p .; Soobpoidy L.T., Mopk T.S., Apbole1 S b. Egu1ygoroeyep 1yegaru. No. \ ν . 1. Meb. 1997; 336 (13): 933938). In practical activities, these drugs revealed a chain of shortcomings: rapid destruction in the body forces a significant increase in the dose and frequency of administration, which causes adverse, including allergic, reactions. This necessitates the search for new dosage forms of erythropoietin to achieve a better biological effect.

These disadvantages can be overcome by chemical modification of the erythropoietin molecule. For this purpose, it is known to use polyethylene glycol (PEG), as well as monomethoxypolyethylene glycol (mPEG), which are neutral polyesters with different molecular weights [Nikitin I.G. Pegylated preparations: current state of the problem and prospects / Nikitin I.G., Storozhakov G.I. // Viral hepatitis: achievements, prospects. 2001. No3 (13). S. 3-8.]

Since PEG and mPEG do not interact directly with polypeptides, various functional groups are introduced into their molecules to obtain the corresponding activated PEG agents | 1 <ox1o \\ kk | A. Oyue1ortey oG Redu1a1eb 1yegGegopk Gog 1ye Tgatey oG Sygois Nerai11k C / 1 <oh1o \\ 'kk | A., Syag1ek 8.A., Natk 1. M. // VyuEgidk. 2001. No. 15 (7). R. 419-429.]. Such PEG agents for erythropoietin pegylation include, in particular, bifunctional PEG derivatives with two terminal aldehyde groups or terminal aldehyde and maleimide groups (UO 2004/081053, publ. 23.09.2004), PEG complex succinamide esters (UO 2001/002017, publ . 11.01.2001; UO 2002/49673, publ. 06/27/2002) and others.

In the application UO 2006/050959 (publ. 18.05.2006) relating to the hematopoietic promoting covalent conjugates of multiple bivalent peptides with hydroxyalkylated starch, the following general indication is given: if the polymer part bearing the bivalent peptide is not capable of directly combining with the peptide, then certain can be used to modify it so that it can react with at least one reactive group of the peptide moiety. For example, it can be modified by introducing a diazo group capable of reacting with the phenolic groups of the peptide.

In the application UO 2006/005667 (publ. 19.01.2006) a method is disclosed for increasing the half-life of a molecule (for example, erythropoietin) in plasma, including covalent binding of this molecule to

- 1 018116 heterocyclic isostere of carboxylic acid (for example, with tetrazole). The description indicates that the spacer linking the molecule to the tetrazole moiety may be oligo (ethylene glycol). In the case where the polypeptides contain one or more tyrosine units, they can be selectively functionalized by azo coupling with the aryldiazonium salt, for example, by treating the tyrosine-containing polypeptide with an aryldiazonium salt with a terminal tetrazole fragment. However, there are no examples of such a conversion in the description of this invention.

As a rule, activated PEG agents interact with the free amino groups of the polypeptides to form a mixture of positional isomers, the number of which does not exceed the number of free amino groups in the protein. It is known that positional isomers of modified proteins have different biological activity (I8 2004/0223950, publ. 11.11. 2004). This creates the prerequisites for expanding the arsenal of such funds due to various options for modifying molecules. For example, changing the positions and methods of attaching PEG to a protein allows one to change the ratios of positional isomers, as well as to obtain new types of conjugates in which, for example, amino groups of polypeptides are not affected.

The use of activated PEG agents containing a paraphenylenediazonium fragment in a molecule for modifying a physiologically active polypeptide, in particular insulin, is disclosed in I8 publication 4179337 (publ. 12/18/1979). The modified polypeptide thus obtained, in particular insulin, has the structure of an amino-derivative formula

where B represents PE6-O-CH ₂ -, PE6-O-CH ₂ -CH (OH) -CH ₂ -O- or PE6-0-C (= 0) -, and represents a peptide chain. The disadvantage of such derivatives is their low stability in the body and the associated possibilities of uncontrolled structural changes and cleavage of reactive particles, in particular diazo compounds.

The closest in technical essence is considered a method for producing a physiologically active water-soluble polypeptide composition from a polypeptide initially having physiological activity, disclosed in 41δ 4179337. This method includes the steps of: (a) reacting at least one terminal carbon atom bearing a hydroxy group, essentially linear a polymer selected from the group consisting of polyethylene glycol and polypropylene glycol having a molecular weight of from 500 to 20,000 Yes, where the polymer is unsubstituted a substituted or substituted alkyl group having less than 5 carbon atoms, with a combining agent to form an activated polymer containing a reactive end group, and (b) the interaction of a physiologically active polypeptide with from 10 to 100 moles of the specified activated polymer by combining the polypeptide with a reactive terminal group of the polymer with the formation of a physiologically active water-soluble polypeptide composition.

In particular, in step (a), paranitrobenzyl chloride is reacted with glycol (PEG with a molecular weight of 500-20,000 Da is suitable) in the presence of alkali in an anhydrous medium, for example tetrahydrofuran, to obtain PEG paranitrobenzyl ether, which is then reduced to the corresponding amine. In the case of PEG benzyl ethers, the reduction of the nitro group is carried out with titanium chloride (111). In the case of esters, catalytic hydrogenation with hydrogen is required to avoid hydrolysis of the ester group. The obtained O-PEG para-aminophenyl ether is diazotized with nitrous acid in an aqueous solution at 0 ° C and combined with a 0.25% insulin solution at 0 ° C for 2 hours; to isolate the desired product, the reaction mixture is dialyzed at 5-10 ° C, which allows to obtain a substantially non-immunogenic modified polypeptide that retains a significant part of the activity of the starting peptide.

In light of the above disadvantages of the modified erythropoietins known in the art, there is a need to develop new compounds and methods for their preparation.

SUMMARY OF THE INVENTION

As a result of the studies, the authors unexpectedly found that high purity monopegylated erythropoietin (EPO) can be effectively obtained by the method, according to which:

a) the free hydroxyl group of monomethoxypolyethylene glycol ¹ - ¹ η where η is an integer in the range from 455 to 1140, acylated with nitrobenzoic acid chlorine or bromohydride in anhydrous pyridine at a temperature of from 10 to 40 ° C and a molar ratio of nitrobenzene benzoic acid chloride of 1 m to 1 : 1 to 1000: 1 to obtain methoxypolyethylene glycol ether of nitrobenzoic acid

- 2 018116

ABOUT

where η takes the above values, which is isolated from the reaction mass by precipitation of a C ₄ -C ₈ simple ether or a C ₆ -C ₈ alkane;

b) the nitro group of methoxypolyethylene glycol ether of nitrobenzoic acid is reduced with a chromium salt (11) in an aqueous or aqueous organic medium with a pH from 1.0 to 6.5 or in an environment of a polar organic solvent miscible unlimitedly with water at a temperature of from 10 to 40 ° С with the production of methoxypolyethylene glycol aminobenzoic acid ester

where η takes the above values, which is isolated from the reaction mass by precipitation of a C ₄ -C ₈ simple ether or a C ₆ -C ₈ alkane;

c) methoxypolyethylene glycol ether of aminobenzoic acid is diazotized with nitrite of an alkali or alkaline earth metal in an aqueous or aqueous organic medium at a temperature of from -2 to 30 ° C or organic nitrite in an environment of a polar organic solvent miscible with water, at a temperature of from -40 to 30 ° С, molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid from 1.1: 1 to 10000: 1 and molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid 3: 1 to 10000: 1 with pic further removing the excess nitrite to obtain an activated PEG agent;

d) the activated PEG agent is introduced into the azo coupling reaction with erythropoietin in an aqueous or aqueous organic medium with a pH from 7.0 to 10.0 and a temperature from 0 to 30 ° C; upon reaching a degree of conversion of at least 70%, the reaction is stopped by adding to a reaction mass of a low molecular weight azo constituent to produce a mixture of monopegylated, dipegylated and unmodified erythropoietin and a blocked PEG agent;

d) the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions to obtain monopegylated erythropoietin.

When implementing the inventive method, an increase in the yield of pegylated EPO and an increase in the circulation time of the pegylated EPO in the bloodstream are achieved with low toxicity of the resulting preparation, which retains a greater proportion of the activity of free EPO compared to the Mirzer drug.

The increase in the yield of pegylated EPO is a consequence of the increase in the total yield of activated pegylating agent due to the production of an intermediate methoxypolyethylene glycol ether of nitrobenzoic acid with a quantitative yield and an intermediate methoxypolyethylene glycol ether of aminobenzoic acid with a yield of 90-100%.

An increase in the circulation time of pegylated EPO in the bloodstream is achieved due to the ability of its tyrosine and histidine units to enter into the azo coupling reaction with active diazo compounds with the formation of biologically active azo derivatives of EPO.

In a preferred embodiment of the invention, in step a), the acylation is carried out at room temperature and diethyl ether is used to precipitate the product.

In another preferred embodiment of the invention, in step b), an acetate buffer solution is used to create and maintain a pH, Cg _{4 is} used as a reducing agent, and diethyl ether is used to precipitate the product.

In another preferred embodiment of the invention, in step c), the diazotization is carried out with sodium nitrite in an aqueous solution of hydrobromic acid, and the excess nitrite is removed with sulfamic acid.

In a further preferred embodiment of the invention, in step c) the diazotization is carried out with tert-butyl nitrite in the presence of HCl in tetrahydrofuran.

In a further preferred embodiment of the invention, in step g) the pH of the reaction mixture is maintained at 9.5 ± 0.3, the degree of conversion of erythropoietin is 80-85%, and tyrosine is used as the low molecular weight azo constituent.

In a further preferred embodiment of the invention, at the stage d), the pH of the reaction mixture is maintained at 7.0 ± 0.3, the degree of conversion of erythropoietin is 70-75%, and histidine is used as a low molecular weight azo constituent.

In a further preferred embodiment of the invention, in step e), the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions from 0.02 to 1.0 M NaCl.

As the chloro or bromohydride of nitrobenzoic acid, any of

- 3 018116 ortho, meta, or paraisomers. Preferred meta and paraisomers, most preferred paraisomers (acid chloride or 4-nitrobenzoic acid bromide anhydride).

The acylation of the free hydroxyl group of monomethoxypolyethylene glycol with chlorobenzoic acid or nitrobenzoic acid bromide is carried out in anhydrous pyridine at a temperature of from 10 to 40 ° C. The molar ratio of nitrobenzoic acid chloro- or bromohydride to monomethoxypolyethylene glycol is from 1: 1 to 1000: 1, preferably from 1: 1 to 10: 1.

The reaction product is isolated from the solution either by pre-evaporation of pyridine and trituration of the residue with a C ₄ -C ₈ simple ether, such as diethyl ether, diisopropyl ether, methyl tert-butyl ether, or precipitation of a C ₄ -C ₈ simple ether from a solution in pyridine, such as diethyl ether, diisopropyl ether, methyl tert-butyl ether or an alkane such as pentane, hexane, heptane, petroleum ether. Diethyl ether is most preferred as ether, and hexane or petroleum ether is most preferred as alkane.

The reduction of monomethoxypolyethylene glycol ether of nitrobenzoic acid to monomethoxypolyethylene glycol ether of aminobenzoic acid is carried out with titanium (III), vanadium (II) or chromium (II) salts, most preferably chromium (II) salts in an aqueous medium at pH from 1.0 to 6.5. Organic acids, for example acetic, citric or tartaric acids, or inorganic acids, for example, hydrochloric, hydrobromic or sulfuric acids, as well as mixtures of organic and / or inorganic acids are used to create and maintain the pH of the aqueous medium. The use of buffer solutions with a pH of from 1.0 to 6.5, more preferably with a pH of from 4 to 6, is preferred. Examples of such solutions are acetate, citrate or tartrate buffer solutions. As reducing agents, acetate, chloride, bromide or chromium sulfate (P) are used. The use of chromium (P) acetate, chloride or sulfate is preferred, more preferably the use of chromium (P) sulfate or acetate.

To ensure the completeness of the course of the reduction reaction, it is necessary that upon its completion a slight excess of a reducing agent is observed, most preferably a chromium salt (P). The completion of the reduction of the nitro compound can be determined visually by the presence of a characteristic coloration of the reaction mass, which does not disappear for at least 5 minutes after the addition of the reducing agent.

The hue depends, in particular, on the nature of the reducing agent and the reagent, ensuring the maintenance of the acidity of the reaction mass, which is known to the average person skilled in the art. For example, when using an acetate buffer solution, an excess of chromium (P) at the beginning of the reaction causes the carmine-red color of the reaction mass, which turns into red-violet during the reduction process and then, in the case of air oxygen, turns into violet.

In the sulfuric acid medium, an excess of chromium (P) causes a blue color, which in the course of reduction turns into greenish-blue and then, in the case of access of atmospheric oxygen, into emerald, slowly turning into violet.

Alternatively, the excess chromium salt (P) can be determined by a qualitative reaction with resorufin

giving violet coloring. The reaction conditions are known to the average person skilled in the art, for example, from the publication Abatek 1., ΒιιζίοΚα E. // M1ktosyesh. Ac1a. 1969. p. 764.

The chromium salts (11) necessary for the reduction are obtained either by dissolving the chromium metal in the corresponding acids, or by reducing the chromium (VI) and / or chromium (III) compounds with metallic zinc or zinc or lead amalgams, preferably metallic zinc, more preferably zinc dust. To avoid oxidation of chromium (P), the resulting solution is stored over several granules of metallic zinc under a layer of a water-immiscible organic solvent, for example petroleum ether, gasoline, kerosene, hexane, benzene, toluene or a mixture thereof, preferably gasoline, kerosene, petroleum ether or hexane, more preferably petroleum ether.

The obtained methoxypolyethylene glycol aminobenzoic acid ester is extracted from the aqueous solution with a halogenated organic solvent such as chloroform, methylene chloride, dichloroethane or a mixture thereof, followed by precipitation with C4-C8 ether or alkane.

Diazotization of methoxypolyethylene glycol ether of aminobenzoic acid is carried out by adding nitrite of an alkali or alkaline earth metal in an acidic aqueous or aqueous organic medium at a temperature of from -2 to 30 ° C. Most preferably, the diazotization temperature in the aqueous medium is in the range of 0 to 5 ° C. The acidic medium in this embodiment is created using organic acids, for example acetic or citric, or inorganic acids, for example, hydrochloric, hydrobromic, sulfuric or phosphoric acids. The molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid is from 1.1: 1 to 1000: 1, preferably from 1.1: 1 to 10: 1. The molar ratio of acid to methoxypolyethylene glycol

- 4 018116 ester of aminobenzoic acid is from 3: 1 to 10000: 1.

The reaction is preferably carried out in the presence of a diazotization catalyst, which is used bromide ions introduced into the reaction mixture in the form of hydrobromic acid or its soluble salts, for example alkali metal bromides. It is most preferable to create an acidic medium with a solution of hydrobromic acid.

Alternatively, this reaction is carried out by acting on an amino compound with organic nitrite in a polar organic solvent, miscibly mixed with water, at a temperature of from -40 to 30 ° C. Most preferably, the diazotization temperature in the organic medium is in the range of −20 to 0 ° C. Preferred organic nitrites are butyl nitrites or amyl nitrites, more preferably tert-butyl nitrite. The molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid is from 1.1: 1 to 1000: 1, preferably from 1.1: 1 to 10: 1.

An acidic medium using a polar organic solvent miscible with water indefinitely is created with solutions of HC1 or HBg in an aliphatic ether, for example diethyl ether, or a cyclic ether, for example dioxane or tetrahydrofuran. The molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid is from 3: 1 to 10000: 1.

Upon completion of diazotization, the activated PEG agent can be stored at reduced temperature for no more than 2 hours in an aqueous or aqueous-organic medium or for no more than 24 hours in a polar organic medium. The term reduced temperature means a temperature of from -2 to 5 ° C in the case of using an aqueous or aqueous-organic medium and from -40 to 0 ° C in the case of using a polar organic medium.

Before using the activated PEG agent for the pegylation of EPO, it is necessary to remove excess nitrite ions, for which urea or sulfamic acid is used. Alternatively, azides of alkali or alkaline earth metals are used.

EPO pegylation is carried out by carrying out the azo coupling reaction of diazotized methoxypolyethylene glycol ether of aminobenzoic acid with EPO in a weakly acidic, neutral or slightly alkaline aqueous or aqueous-organic medium at a temperature of from 0 to 30 ° C.

The pH range for pegylation is selected depending on the preferred positions of the addition of the PEG agent to EPO. In a slightly acidic and neutral medium (pH 6.0-8.0), pegylation occurs predominantly on the amino acid residues of histidine. In this embodiment, it is preferable to establish and stabilize the pH using a phosphate, acetate or citrate buffer solution. In a slightly alkaline medium (pH 8.0-10.0), pegylation occurs both on the amino acid residues of histidine and tyrosine. In this embodiment, it is preferable to establish and stabilize the pH using an acetate, borate or borate-carbonate buffer solution. The molar ratio of the diazotized methoxypolyethylene glycol ester of 4-aminobenzoic acid to EPO is from 1: 1 to 20: 1, most preferably from 3: 1 to 8: 1.

The pegylation process is monitored by size exclusion or reverse phase HPLC. Upon reaching the degree of conversion of EPO, at least 70% of the pegylation reaction is stopped by adding to the reaction mass a low molecular weight azo component, which is used substances of phenolic nature or their esters, substances having the nature of aromatic amines or substances having a heterocyclic nature, in which the heterocycle is able to act as an azo bearing in the azo coupling reaction. Most preferred are tyrosine and histidine.

The isolation of monopegulated EPO from the reaction mixture is carried out by conventional methods of ion exchange chromatography, sequentially using buffer solutions with increasing ionic strength. Protein concentration is determined by HPLC or spectrophotometric using an A ₂₈₀ value of 0.743 for a solution with an EPO concentration of 1 mg / ml.

Further, the invention will be illustrated by the following examples, confirming the possibility of its implementation with the achievement of the technical result indicated in the description.

Example 1. Monomethoxypolyethylene glycol ether of 4-nitrobenzoic acid (M.M. 30 kDa).

To a solution of 30 g (1 mmol) of monomethoxypolyethylene glycol (M.M. 30 kDa) in 70 ml of anhydrous pyridine, 1.9 g (10 mmol) of 4-nitrobenzoyl chloride are added and stirred for 30 minutes at room temperature. Next, the reaction mass is evaporated on a rotary evaporator to half the initial volume and the reaction product is precipitated by adding five times the volume of diethyl ether. The precipitate was filtered off with suction, washed several times with diethyl ether and dried in vacuo. Yield 30 g (100%).

Example 2. Monomethoxypolyethylene glycol ether of 4-aminobenzoic acid (M.M. 30 kDa).

A) Preparation of a solution of chromium sulfate (11).

To 100 ml of a 5% solution of K ₂ Cr ₂ O ₇ - zinc dust is added in portions of about 0.5 g in 20% H ₂ §0 ₄ until the reaction mass is green, after which 30 ml of petroleum ether is added to the reaction mass and continue to be added zinc dust before the reaction mass acquires a blue color. During the recovery process, the medium is kept acidic. The solution is allowed to settle, after

- 5 018116 which is poured into a storage vessel, several granules of metallic zinc are added and if more petroleum ether is required, they are equipped with a water seal and left overnight. The next day, the dark blue solution of chromium sulfate (II) St§0 _{4 is} completely ready for use as a reducing agent and remains suitable for this for about 1 month.

B) Reduction of 4-nitrobenzoic acid monomethoxypolyethylene glycol ether.

To a solution of 3.0 g (0.1 mmol) of 4-nitrobenzoic acid monomethoxypolyethylene glycol ester (M.M. 30 kDa) obtained in accordance with Example 1, an excess of chromium sulfate is added in 20 ml of acetate buffer with pH 6 (11), the reaction mixture immediately acquires a carmine-red color, the vessel is tightly closed with a lid and stirred for 5 minutes, while the red tint should remain. Next, the aqueous phase is extracted five times with equal portions of methylene chloride (about 30 ml each). The organic extracts are combined and evaporated under vacuum on a rotary evaporator at 40 ° C to 1/5 of the original volume, after which the resulting 4-aminobenzoic acid monomethoxypolyethylene glycol ether is precipitated with diethyl ether, filtered off, washed with diethyl ether and dried in vacuo. Yield 2.8 g (93%).

Example 3. Diazotized monomethoxypolyethylene glycol ester of 4-aminobenzoic acid (activated PEG agent with a molecular weight of 30 kDa).

To a solution cooled to 1 ° C of 0.077 g (2.5 μmol) of 4-aminobenzoic acid monomethoxypolyethylene glycol ether (M.M. 30 kDa) obtained in accordance with Example 2, 1 ml of chilled 0 is added in 1 ml of 0.1 M HBg , 01 M ΝαΝΟ ₂ and kept in an ice bath for 20 minutes, after which 1 ml of 0.01 M ΝΗ ₂ 8Ο ₃ Η is poured and immediately used for pegylation or stored in the refrigerator for no more than 2 hours.

ESP (water, pH 4) λ, nm (1d ε): 203 (4.86), 256 (4.51), 275 (4.43, shoulder).

Example 4. Diazotized monomethoxypolyethylene glycol ether of 4-aminobenzoic acid (activated PEG agent with a molecular weight of 30 kDa).

To a solution cooled to -35 ° C, 0.077 g (2.5 μmol) of 4-aminobenzoic acid monomethoxypolyethylene glycol ether (M.M. 30 kDa) obtained in accordance with Example 2 in 0.5 ml of a 0.2 M HC1 solution in tetrahydrofuran is added 0.5 ml of 0.01 M tert-butyl nitrite in tetrahydrofuran and left at -35 ° C for at least 30 minutes (but not more than 24 hours), after which 0.5 ml of 0.01 M ΝΗ are added ₂ 8Ο ₃ Η and immediately used for pegylation.

Example 5. Obtaining monopegylated EPO in a slightly alkaline environment.

To a solution of 10 mg (0.5 μmol) EPO cooled to 3 ° C in a borate-carbonate buffer with a pH of 9.5, a chilled solution of 0.077 g (2.5 μmol) of diazotized 4-aminobenzoic acid diamerized monomethoxypolyethylene glycol ether is added (M.M. 30 kDa) obtained in accordance with example 3 or 4, maintaining the pH of the reaction mixture 9.5 ± 0.3. The reaction mixture was stirred for approximately 3 hours while cooling, monitoring the progress of reverse-phase HPLC transformations (Whooshavy 300-5C4, 250x4.6 mm column, spectrophotometric detection at 220, 280, 340 and 400 nm, gradient elution: from 30% aq. Acetonitrile + 0.2% TFA to 80% aq. Acetonitrile + 0.2% TFA). Upon reaching the degree of conversion of EPO equal to 80-85%, a tyrosine solution is poured, stirred for 5 minutes and the pH is adjusted to 5.0-6.5 with acetic acid. Next, the reaction mixture, containing a mixture of dipipegylated, mono-pegylated and unmodified EPO, as well as a blocked pegylating agent, is separated by ion exchange chromatography with an increase in the ionic strength of buffer elution solutions from 0.02 to 1.0 M No. C1. The yield of purified mono-pegylated EPO is 4.2 mg (42%, counting on EPO).

Example 6. Obtaining monopegylated EPO in a neutral environment.

A cooled solution of 0.077 g (2.5 μmol) of diazotized 4-aminobenzoic acid diamerized monomethoxypolyethylene glycol ether (M.M. 30 kDa) is added to a solution of 10 mg (0.5 μmol) EPO cooled to 1 ° C. obtained in accordance with example 3 or 4, maintaining the pH of the reaction mixture 7.0 ± 0.3. The reaction mixture was stirred for about 5 hours while cooling, monitoring the progress of reverse-phase HPLC transformations (SomershavI 300-5C4 column, 250x4.6 mm, spectrophotometric detection at 220, 280, 340 and 400 nm, gradient elution: from 30% aq. Acetonitrile + 0.2% TFA to 80% aq. Acetonitrile + 0.2% TFA). Upon reaching the degree of conversion of EPO equal to 70-75%, a histidine solution is poured, stirred for 5 minutes and the pH is adjusted to 5.0-6.5 with acetic acid. Next, the reaction mixture, containing a mixture of dipipegylated, mono-pegylated and unmodified EPO, as well as a blocked pegylating agent, is separated by ion exchange chromatography with an increase in the ionic strength of buffer elution solutions from 0.02 to 1.0 M No. C1. The yield of purified mono-pegylated EPO is 3.8 mg (38%, counting on EPO).

Example 7. The toxicity study of the covalent conjugate of polyethylene glycol with erythropoietin.

Conjugate of polyethylene glycol with erythropoietin in the dose range from 50 to 500 IU / kg is administered to male CBA mice. Animals are counted daily, examined for 5 days, and blood is collected for analysis. On the fifth day, animals are euthanized to study the condition of internal organs.

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No lethal outcomes were noted during the study. The results obtained indicate the absence of significant changes in hematological and biochemical blood parameters, as well as pathological changes in the internal organs of mice. According to the results of studies, the conjugate obtained by the method in accordance with the invention is non-toxic to humans with a single dose of up to 500 IU / kg.

Example 8. The study of the pharmacokinetics of the covalent conjugate of polyethylene glycol with erythropoietin.

Male mice of CBA mice were injected intramuscularly with 1 μg of a conjugate of polyethylene glycol with erythropoietin, after which blood was collected on the first day 2 hours after injection and then every 24 hours for 10 days. Blood samples were incubated for 45 min at 37 ° C, after which the thrombus was separated and re-incubated at 4 ° C, the resulting serum was centrifuged and stored at -65 ° C until testing. The content of polyethylene glycol with erythropoietin in blood serum samples was determined by immuno-enzyme analysis of Vusheps EPO EY8A (Vusheps, USA). The obtained comparative data on the pharmacokinetics of free EPO, Mircera drug and conjugate obtained by the method in accordance with the invention are presented in table. one.

Table 1. Pharmacokinetics of free EPO, Mircera drug and conjugate obtained by the method in accordance with the invention

Day Serum concentrations, IU / ml free EPO the drug "Mircera" conjugate one 2850 900 850 2 2800 1500 1200 3 2404 2911 3300 4 1814 3713 4050 5 901 3600 4100 6 536 3424 3820 7 200 3000 3100 8 10 2000 1700 nine NGOs 1200 1300 10 NGOs 700 850

NGO - below the limit of determination by enzyme immunoassay.

Example 9. The study of the specific activity of the covalent conjugate of polyethylene glycol with erythropoietin.

The specific activity of the conjugate of polyethylene glycol with erythropoietin is evaluated by the increase in the number of red blood cells after its parenteral administration to normocytemic mice of the B _& E ₂ T _{1 line} , aged 7 to 15 weeks. Animals are given a dose that causes a measurable increase in the number of reticulocytes. Counting the number of reticulocytes in whole blood is carried out by microfluorimetry in a flow cytometer. The activity of the studied samples ίη νίνο is calculated by the parallel line method in comparison with the biological activity of the standard sample. Indicators of biological activity are given in table. 2.

Table 2. The biological activity of free EPO, Mircera drug and conjugate obtained by the method in accordance with the invention

A drug The specific activity, IU / mg % activity Free EPO 100-10 ³ one hundred Conjugate 17-th ³ 24 Mircera 14

Research data shows that the conjugate obtained by the method in accordance with the invention retains a significant portion of the biological activity of unmodified EPO and is superior in activity to Mircera.

Claims (8)

CLAIM 1. The method of obtaining monopegylated erythropoietin, characterized in that: a) the free hydroxyl group of monomethoxypolyethylene glycol where η is an integer in the range from 455 to 1140, acylated with nitrobenzoic acid chloride or bromohydride in anhydrous pyridine at a temperature of from 10 to 40 ° C and a molar ratio of nitrobenzoic acid chloride from monomethoxypol to 1 monomethoxy lipol: 1 : 1 to obtain methoxypolyethylene glycol ether of nitrobenzoic acid where η takes the above values, which is isolated from the reaction mass by precipitation of a C ₄ -C ₈ simple ether or a C ₆ -C ₈ alkane; b) the nitro group of methoxypolyethylene glycol ether of nitrobenzoic acid is reduced with a chromium salt (11) in an aqueous or aqueous-organic medium with a pH from 1.0 to 6.5 or in an environment of a polar organic solvent miscible unlimitedly with water at a temperature of from 10 to 40 ° С with obtaining methoxypolyethylene glycol ester of aminobenzoic acid where η takes the above values, which is isolated from the reaction mass by precipitation of a C ₄ -C ₈ simple ether or C ₆ -C ₈ alkane; c) methoxypolyethylene glycol ether of aminobenzoic acid is diazotized with nitrite of an alkali or alkaline earth metal in an aqueous or aqueous organic medium at a temperature of from -2 to 30 ° C or organic nitrite in an environment of a polar organic solvent miscible with water, at a temperature of from -40 to 30 ° C, the molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid from 1.1: 1 to 10000: 1 and the molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid from 3: 1 to 10000: 1 s osleduyuschim removing excess nitrite to give an activated PEG agent; d) the activated PEG agent is introduced into the azo coupling reaction with erythropoietin in an aqueous or aqueous organic medium with a pH of 7.0 to 10.0 at a temperature of 0 to 30 ° C; upon reaching a degree of conversion of at least 70%, the reaction is stopped by adding to the reaction mass of a low molecular weight azo component to produce a mixture of monopegylated, dipEGylated and unmodified erythropoietin and a blocked PEG agent; d) the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions to obtain monopegylated erythropoietin. 2. The method according to claim 1, characterized in that in stage a) the acylation is carried out at room temperature and diethyl ether is used to precipitate the product. 3. The method according to claim 1 or 2, characterized in that in step b) an acetate buffer solution is used to create and maintain the pH, Cr ₄ 0 is used as a reducing agent, and diethyl ether is used to precipitate the product. 4. The method according to any one of claims 1 to 3, characterized in that in step c) the diazotization is carried out with sodium nitrite in an aqueous solution of hydrobromic acid, and the excess nitrite is removed with sulfamic acid. 5. The method according to any one of claims 1 to 3, characterized in that in step c) the diazotization is carried out with tert-butyl nitrite in the presence of HCl in tetrahydrofuran. 6. The method according to any one of claims 1 to 5, characterized in that in step g) the pH of the reaction mixture is maintained at 9.5 ± 0.3, the degree of conversion of erythropoietin is 80-85%, and tyrosine is used as a low molecular weight azo component. 7. The method according to any one of claims 1 to 5, characterized in that at the stage d) the pH of the reaction mixture is maintained at 7.0 ± 0.3, the degree of conversion of erythropoietin is 70-75%, and histidine is used as a low molecular weight azo component. 8. The method according to any one of claims 1 to 7, characterized in that in step d) the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer eluting solutions from 0.02 to 1.0 M IaS1.