EA020220B1 - Covalent conjugate of polyethyleneglycol with polypeptide having interferon-gamma activity - Google Patents

Abstract

The invention relates to medicine and biotechnology and describes a covalent conjugate of polyethyleneglycol with polypeptides, having a biologic activity of interferone-gamma (IFN-γ) of formula (I)where n is an integer from 100 to 1200, A is an amino acid fragment of tyrosine or histidine, PP is a polypeptide with IFN-γ activity, and a method of producing it. The technical result is aimed at expanding interferon spectrum and increasing circulation time in blood flow, preserving a substantial biologic activity of an unmodified polypeptide.

Description

FIELD OF THE INVENTION

The invention relates to medicine and biotechnology and relates to a covalent conjugate of polyethylene glycol with polypeptides having the biological activity of interferon-gamma (ΙΕΝ-γ), in which the polymer is attached to the polypeptide via the azo group.

Background of the Related Art

Interferon-gamma (ΙΕΝ-γ) is a dimerized soluble cytokine with a molecular weight of about 17 kDa and the only representative of type II interferons [Sgau R. ^., Sosys1 Ό.ν. 81gis1ig about! (Ns Pitan 1thtii yKsGGsgop depe. No. 1 (ig. Νοί. 298 (5877) (Aidiz1 1982). P. 859-863].) In contrast to interferon-α and interferon-β, which can be expressed by all cells, ΙΕΝ-γ produced only by TH1, T and-cells.

ΙΕΝ-γ is necessary for innate and adaptive immunity to viral and intracellular bacterial infections, as well as for tumor control. The role of ΙΕΝ-γ in the immune system consists, in particular, in its ability to suppress the replication of the virus as such. But the main activities of γ-γ are immunostimulatory and immunomodulatory activities [8cNoeybot 1.K., Yozyo S.V. Kediayop oG yKegGegop-datta yippd shia (eai ayaryue 1 type gesropez. Λάν. Ittiηο1. ^ 1. 96 (2007). P. 41-101]. Aberrant expression of ΙΕΝ-γ is associated with a number of inflammatory and autoimmune diseases.

Interferon-gamma is used to treat chronic granulomatous diseases [Toy R.A., Soa K. ShegGegop Datta-1b. And he \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ following Reshagas with odo 1 Negareis ro! Bigids. ^ 1. 43 (1) (Gapiagu 1992). RR 111-122], osteoporosis [Keu b. L., K1ez \ ν.Ε. Koypdth K.M., Na1sNeg N.S. KesotlagK Nitap and KegGegop Datta (Negaru Gogz (Eore (Goz1z. 1.Resha (g. ^ 1. 121 (1)) (Mu 1992), R. 119].

In the complex therapy of chronic viral hepatitis B and C, HIV / AIDS infection, tuberculosis, cancer, urogenital chlamydia and genital herpes virus infection, the drug Ingaron (manufacturer - NSh1 Pharmaclon, RF), which is a lyophilizate of recombinant human interferon-gamma, is used with a molecular weight of 16.9 kDa, consisting of 144 amino acid residues, at the beginning of the sequence of which the first three Souz-Tug-Suz are replaced by Me1. The preparation was obtained by microbiological synthesis in a recombinant strain of Escherichia coli and purified by column chromatography.

Nevertheless, the use of ΙΕΝ-γ preparations in medicine is limited by a number of factors, among which the most significant is the lack of enzymatic stability in the body | Sap (e11 K. e (a1. Rnaghasoxiconium sylvilinus). 1.! N (egGegop Kez. ^ 1.6 (1986). P.671-675; N. N. N., Sep (C., 1. V. V., Sagoya S., Knightapp N.V., NosNiN E. Koel eG ( Not sarboku (ergta1 zeppse oG (Hebtuuu1as (tv ^ (u oG Nitap 1tip 1n (erGegop. 1. B1cNp1. ^ 1.7 (1988). R.199216; KshepP'ap / Ι., Vaeg A., KpnNp. RNagtasocggice s (pyu oG Nrozote-eparsi1a (her Nitap t1egG gopdatta ayeg t (ha \ 'epoiz apy tyatisi1ag t] es (juh pis. 1. ShegGegop Kez. ^ 1. 10 (3) (1st type 1990). P. 3337341], which leads to the need to increase the dose and frequency of administration of the drug , which, in some cases, is associated with side effects, such as allergies. For example, the drug Ingaron is administered daily or every other day. In this connection, there is a need for chemical modification of the interferon-gamma molecule in order to overcome these disadvantages.

One of the ways is to change the amino acid sequence and obtain more stable polypeptides with ΙΕΝ-γ activity. In patent KI 2326944 (published on June 20, 2008), a recombinant human ΙΕΝ-γ analogue is proposed - deltaferon with a molecular weight of 16.2 kDa, which has a deletion of 10 amino acid residues at the C-terminus of the molecule and replacement of the CCCK cluster with KC8A compared to the natural ΙΕΝ -γ. As a result of this modification, the молекулы-γ analog molecule (deltaferon) appeared to be resistant to proteolysis.

In another case, for the modification of various polypeptides it is known to use monomethoxypolyethylene glycol (mPEG), which is a neutral polyester with different molecular weights [I. Nikitin, T. N. Storozhakov Pegylated drugs: current state of the problem and prospects, in the collection Viral hepatitis: achievements of the perspective. No. 3 (13) (2001). S. 3-8].

As a rule, in covalent conjugates, the polymer fragment is attached to the polypeptide via one of the free amino groups of the latter [Ko / 1o ^ sk1 A., CHag1ez 8.A., Natz EM. Oeteortep (oG Redu1a (her Iη (e ^ Te ^ οηz Gog (Ne Tgea (tep (oG SNgoshs Nerayz S. VyuBgidz .; ^ 1. 15 (7) (2001). P. 419-429).

In patent I8 5109120 (publ. 28.04.1992) a recombinant ΙΝΕ-γ conjugate is proposed in which a polyethylene glycol fragment is attached to the polypeptide via the Ν-terminal amino group or the terminal amino group of one of the lysine fragments.

Such preparations are mixtures of positional isomers, the chemical structure of which differs at the point of attachment of the polymer to the polypeptide. Theoretically, the number of positional isomers in this case is equal to the number of free amino groups in the polypeptide [Blokhin NP, Nikitin IG Features of pharmacological dynamics and kinetics of pegylated α-interferon (40 kDa)

- 1,020,220

Pegasis: new possibilities for the treatment of chronic hepatitis C in the collection Materials of the VII Russian Conference Hepatology today. RZHGGK. 2002. S. 6.].

It is known that positional isomers of polypeptides 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 protein molecules. For example, a change in 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.

Patent I8 6958388 (publ. 25.10.2005) and international application AO 2002/081507 (publ. 17.10.2002) disclose polypeptide variants of interferon-gamma having the ability to bind to receptor ΙΡΝΟ containing from 1 to 10 modifications of its amino acids, preferably introduction of a cysteine unit covalently attached to a non-polypeptide fragment, which is, for example, linear or branched polyethylene glycol, with orthopyridyldisulfide (PEG-OR88) and maleimide (PEG-ML) PEG derivatives being used as pegylating agents.

When PEG-OR88 is used in the resulting conjugate, the polymer is bound to the cysteine fragment of the polypeptide by an unstable disulfide bond, and when PEG-ML is used, the polymer binds to both thiol and amino groups of the polypeptides, forming a mixture of the corresponding positional isomers [Gershkovich AA, Kibirev V .TO. Chemical synthesis of peptides. Kiev, Naukova Dumka. 1992; 8 Nap 8. Aopd. This is a great deal! SHS Rgs55. 1ps 1993].

In the patent I8 179337 (publ. 12/18/1979) physiologically active insulin conjugates are disclosed containing a para-phenylenediazonium fragment in the molecule and having the structure of an amino derivative formula

where B represents REO-O-CH ₂ -, REO-O-CH ₂ -CH (OH) -CH ₂ -O- or REO-O-C (= O) -, and represents a peptide chain that can be considered as nearest analogues.

The disadvantages of such derivatives are the relatively large number of possible variants of positional isomers due to the addition of the polymer part to the amino groups of the polypeptides and their low stability in the body and, as a result, the possibility of uncontrolled structural changes and cleavage of reactive particles, in particular, diazocompounds.

Thus, there is a need for new conjugates of polypeptides with ΙΓΝ-γ activity.

SUMMARY OF THE INVENTION

The inventors unexpectedly found that the disadvantages of the prior art can be overcome by synthesizing monopegylated polypeptides with ΙΓΝ-γ activity having the general structure (I)

where n is an integer in the range from 100 to 1200,

A is an amino acid fragment of tyrosine or histidine,

PP is a polypeptide with ΙΓΝ-γ activity, in which the polymer is attached to the tyrosine or histidine fragments of the polypeptide via the azo group.

The technical result of this modification of the chemical structure is to increase the duration of circulation of the active substance in the blood while maintaining a significant part of the biological activity of the unmodified polypeptide.

According to the invention, interferon-gamma (ΙΝΓ-γ) or deltaferon can be used as a polypeptide with ΙΓΝ-γ activity.

- 2,020,220

Thus, the first object of the invention is a covalent conjugate of polyethylene glycol with a polypeptide having interferon-gamma (ΙΕΝ-γ) activity of the formula (I)

where η is an integer in the range from 100 to 1200,

A is an amino acid fragment of tyrosine or histidine,

PP is a polypeptide with ΙΕΝ-γ activity.

The covalent conjugate is prepared by azo coupling of 4 - ([methoxypolyethylene glycol] carbonyl) benzenediazonium (M.M. 30 kDa) with histidine and tyrosine fragments of polypeptides acting as azo constituents in this reaction.

In order to prove the structure of the conjugate as an azo compound, in particular, electronic absorption spectroscopy (ESP) is used, the spectral characteristics (intensity, width and position of the absorption band maxima) of solutions of the initial and modified polypeptides with ΙΕΝ-γ activity are compared and the obtained patterns are interpreted from the perspective of the theory of organic color compounds, optionally, using adequate methods of quantum chemical modeling. The characteristics of the maxima of the absorption bands are given in table. one.

Table 1. Characteristics of the maxima of the absorption bands of ΙΕΝ-γ, deltaferon, and their mono-pegylated analogs (water, pH 6.5, 1 1.0 cm)

Compound Characteristic Value ΙΓΝ-Υ NM 219 280 - - p1% Wasp 86.2 9.7 - - PEG-ΙΓΝ-γ η = 670-630 λ-max / nm 224 281 344 414 * C-1% ^ 1cm 75.0 10,2 2.7 3.0 Deltaferon Aimake g NM 205 280 - - * 1cm 83.1 σ. . oo ___________________________________________ ί - - PEG-deltaferon η = 670-680 Amane / NM 208 278 346 412 * p |% ^£ 1cm 71.7 9.3 2.9 3,1

g-1% ^{| ck} - absorption of 1% (w / v) solution at 1 1 cm * - manifests itself in the form of a shoulder.

The appearance of absorption bands in monopegylated ΙΕΝ-γ and deltaferon with maxima at 344.414 and 346.412 nm can be explained, for example, from the standpoint of the color theory of organic compounds, local electronic transitions in chromophore systems not conjugated by the conjugation of diarylazo groups, ΙΕΝ-γ formed as a result of covalent addition of PEG -agent to tyrosine and histidine units of polypeptide molecules. The broadening of the bands is due to the presence of several positional isomers with close energies of electronic-vibrational transitions.

The achievement of the technical result, which consists in increasing the circulation time in the bloodstream of covalent conjugates (pegylated polypeptides) with ΙΕΝ-γ activity, is confirmed in a series of studies on a mouse model. The results of comparative studies of ίη νίνο are given in table. 2.

- 3,020,220

Table 2. Relative concentrations of ΙΤΝ-γ and deltaferon and their conjugates in accordance with the invention in the blood of mice

Day Relative concentration (a% of maximum) ΙΓΝ-Υ PEG-ΙΕΝ-γ η = 670-680 Deltaferon PEG-Deltaferon η = 670-680 one 100, 0 48.5 100, 0 39, Θ 2 2,3 one hundred 6.1 one hundred 3 0 28.7 0 42.3 4 0 6.4 0 11, 6 5 0 1,2 0 4.1 ⁶ 0 0 0 0

The data obtained show that the conjugates of ΙΤΝ-γ and deltaferon in accordance with the invention have a longer circulation time in the bloodstream compared to non-pegylated polypeptides.

The achievement of the technical result, which consists in the conservation by covalent conjugates of a significant part of the biological activity of the starting polypeptides, is confirmed by the results of determining the residual activity of pegylated polypeptides with ΙΝΤ-γ activity.

Table 3. The activity of ΙΝΤ-γ, deltaferon and their conjugates (residual activity)

A drug Activity polypeptide, IU / mg % residual polypeptide activity ΪΝΕ-γ 2.010 ⁷ - PEG-ΙΝΕ-γ η = 670-680 3.4-10 ⁶ 17% Deltaferon 7.9-10 * - PEG-Deltaferon η = 670-680 1.810 ⁶ 23%

The covalent conjugate of gamma polyethylene glycol (ΙΤΝ-γ) of formula (I) with a polypeptide having an interferon activity

where η is an integer in the range from 100 to 1200,

A is an amino acid fragment of tyrosine or histidine,

PP is a polypeptide with ΙΤΝ-γ activity, can be obtained by a method including the stages at which:

a) methoxypolyethylene glycol aminobenzoic acid ester

where η takes values from 100 to 1200, 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, unlimitedly miscible with water, at a temperature of -40 to 30 ° C, the molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid is from 1.1: 1 to 1000: 1 and the molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid 3: 1 to 10000: 1, followed by removal of excess of nitrite and obtaining the activated pegylating agent - 4 - ([methoxypolyethylene glycoloxy] carbonyl) benzenediazonium

- 4 020220 where η - takes the above values;

b) an activated pegylating agent without isolation from the reaction mixture is introduced into the azo coupling reaction with a polypeptide having ΙΝΕ-γ activity in an aqueous or aqueous-organic medium with a pH from 7.0 to 10.0 at a temperature from 0 to 30 ° C; upon reaching the degree of conversion of at least 70%, the reaction is stopped by adding a low molecular weight azo component to the reaction mass to obtain a mixture of a mono-pegylated, di-pegylated and unmodified polypeptide with gamma interferon activity and a blocked PEG agent, and

c) the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions to obtain a mono-pegylated polypeptide having ΙΝΕ-γ activity.

When implementing this method, an increase in the yield of pegylated polypeptide having активность-γ activity and an expansion of the arsenal of drugs for combating viral and some bacterial infections are provided.

An increase in the yield of the pegylated polypeptide having ΙΝΕ-γ activity is achieved by quantitatively converting the PEG agent into the active form immediately before conjugation, excluding the degradation of the active moiety — the diazo group during transportation and storage.

An increase in the circulation time of the pegylated polypeptide with ΙΝΕ-γ activity in the bloodstream is possible due to the ability of tyrosine and histidine ΙΝΕ-γ units to react with azo compounds with active diazocompounds to form biologically active azo derivatives of polypeptides with ΙΝΕ-γ activity.

If necessary, increasing the stability of the pegylating agent during storage is achieved by using organic nitrite for activation of the pegylating agent in a polar organic solvent that mixes unlimitedly with water, which additionally allows expanding the temperature range of the diazotization reaction.

In a preferred embodiment of the invention, in step a), 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 a) 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 b), a borate-carbonate buffer solution is used to create and maintain the pH, the degree of conversion of polypeptides with ΙΝΕ-γ activity is 80-85%, and tyrosine is used as a low molecular weight azo-component.

In a further preferred embodiment of the invention, in step c), 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 No. C1.

At the stage a) 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.

In the first embodiment of diazotization, alkali or alkaline earth metal nitrite is used in an acidic aqueous or aqueous-organic medium. The most preferred diazotization temperature range is from 0 to 5 ° C. An acidic medium is created using organic acids, for example, with acetic acid or its halogen derivatives, such as chloroacetic, trichloroacetic, bromoacetic, tribromoacetic, trifluoroacetic acids, as well as citric or tartaric acids, or inorganic acids, for example, hydrochloric, hydrobromic , sulfuric or phosphoric acids, as well as a mixture of organic and / or inorganic 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 ether 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.

In the second embodiment, diazotization is carried out using organic nitrite in an environment of a polar organic solvent, unlimitedly miscible with water, at a temperature of from -40 to 30 ° C. The most preferred diazotization temperature range is from -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 in a polar organic medium is created with solutions of HC1 or HBr 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 no more than 24 hours in a polar

- 5,020,220 in an organic environment without significant loss of its ability to azo combination. 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 pegylation of interferon, it is necessary to remove excess nitrite ions, for which urea or sulfamic acid is added to its solution. Alternatively, azides of alkali or alkaline earth metals are used.

At stage b), pegylation of a polypeptide with β-γ activity is achieved as a result of the reaction of azo coupling of a diazotized methoxypolyethylene glycol ester of aminobenzoic acid with interferon a-2b in a neutral or slightly alkaline aqueous or aqueous-organic medium at a temperature of from 0 to 30 ° С. The most preferred pH range for pegylation is from 9 to 10. The pH is maintained using a suitable buffer solution, for example a borate-carbonate buffer solution. The choice of solution is within the competence of the average person skilled in the art. The molar ratio of the diazotized methoxypolyethylene glycol ester of 4-aminobenzoic acid to the polypeptide with ΙΝΤ-γ activity 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 desired degree of conversion of the polypeptide, the pegylation reaction is stopped by adding a low molecular weight azo moiety to the reaction mass. For this purpose, phenolic substances or their esters, substances having the nature of aromatic amines, or substances having a heterocyclic nature, in which the heterocycle is capable of acting as an azo bearing in the azo coupling reaction, are used as low molecular weight azo-constituent. Tyrosine and histidine are most preferred, more preferably tyrosine. Preferably, the degree of conversion of the polypeptide calculated by the results of HPLC analysis is 80-90%.

The isolation of a mono-pegylated polypeptide with ΙΝΤ-γ activity from the reaction mixture is carried out by conventional methods of ion exchange chromatography, sequentially using buffer solutions with increasing ionic strength. The concentration of the polypeptide with β-γ activity is determined by HPLC or spectrophotometrically using the corresponding A ₂₈₀ value for a solution with a polypeptide concentration of 1 mg / ml.

Information confirming the possibility of carrying out the invention

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

To a solution of 0.077 g (2.5 μmol) of 4-aminobenzoic acid monomethoxypolyethylene glycol ether (m.m. 30 kDa) in 1 ml of 0.1 M HBg, 1 ml of chilled 0.01 M Ν αΝ _{2 is} added to a solution cooled to 1 ° C and kept in an ice bath for 20 minutes, after which 1 ml of 0.01 M Η ₂ Ν 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 (1§ ε): 203 (4.86), 256 (4.51), 275 (4.43, shoulder).

Example 2. 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 in 0.5 ml of a 0.2 M solution of HC1 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 no more than 24 hours), after which 0.5 ml of 0.01 M Η ₂ Ν 8Ο ₃ Η are added and immediately used for pegylation .

Example 3a Obtaining monopegylated polypeptide having the activity of ΙΝΤ-γ (interferon-gamma).

A cooled solution of 0.077 g (2.5 μmol) of diazotized 4-aminobenzoic acid diazotised monomethoxypolyethylene glycol ester (m.m .30 kDa) obtained in accordance with example 1 or 2, maintaining the pH of the reaction mixture 9.5 ± 0.3. The reaction mixture was stirred for about 3 hours while cooling, monitoring the progress of reverse-phase HPLC transformations (Kotoshaky 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 the corresponding polypeptide 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 dipegylated, mono-pegylated and unmodified polypeptide with β-γ activity, as well as a blocked pegylating agent, are separated by ion exchange chromatography with an increase in the ionic strength of buffer elution solutions from 0.02 to 1.0 M NaCl.

- 6,020,220

The yield of purified monopegylated ΙΕΝ-γ is 44% (counting on ΙΕΝ-γ).

ESP (water, pH 6.5, 1 1.0 cm), X _max , nm (E ¹ % _{1 cm} ): 224 (75.0), 281 (10.2), 344 (2.7), 414 (3.0).

Example 3b Obtaining monopegylated polypeptide having the activity of ΙΝΕ-γ (deltaferon).

Under the conditions described in example 3 a, a purified monopegulated deltaferon is obtained with a yield of 51% (counting on deltaferon).

ESP (water, pH 6.5, 1 1.0 cm), X _max , nm (E ^1% ! _Cm ): 208 (71.7), 278 (9.3), 346 (2.9), 412 (3.1).

Example 4. Determination of the circulation time of monopegylated ΙΕΝ-γ and deltaferon in the blood in a mouse model.

Male mice of CBA line are intramuscularly injected with 1 μg of pegylated polypeptide with ΙΕΝ-γ activity in accordance with the invention (30 kDa), after which blood is collected on the first day 2 hours after injection and then every 24 hours for 10 days. As a control, the corresponding unmodified polypeptide is used, which is introduced according to the same scheme. Blood samples are incubated for 45 min at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the resulting serum is centrifuged and stored at -65 ° C until testing. The content of polypeptides (PG / ml) in blood serum is determined using enzyme-linked immunosorbent assay, the results are averaged. The relative polypeptide content (in%) is calculated, while the average maximum concentration of protein in the blood in each group of animals is taken as 100%.

Example 5. Determination of the activity of monopegylated and unmodified ΙΕΝ-γ and deltaferon.

Antiviral activity is determined by micromethod in 96-well flat-bottom plates to suppress the cytopathic effect of the test virus in the diploid culture of L-68 cells (200,000 cells / ml). To do this, use the EMC strain Columbia strain virus at a dose of 100 CPD ₅₀ and the nutrient medium Needle MEM with the addition of 10% cattle serum. The residual activity of the conjugates is expressed with respect to the results of parallel titration of the activity of a standard solution of the corresponding polypeptide having the biological activity of ΙΕΝ-γ.

Claims (1)

CLAIM A covalent conjugate of polyethylene glycol with a polypeptide having an interferonagamma activity (ΙΕΝ-γ) of the formula (Ι) (I) where η is an integer in the range from 100 to 1200; A is the amino acid fragment of tyrosine or histidine; PP is a polypeptide with ΙΕΝ-γ activity selected from the group consisting of ΙΕΝ-γ and deltaferon. About Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2