EA020257B1 - THE NEW STABLE POLYETHYLENE GLYCOL CONJUGATE OF INTERFERON ALPHA, REPRESENTED BY ONE POSITIONAL ISOMER PEF-NαH-IFN AND IMMUNOBIOLOGIC PREPARATION BASED THEREON - Google Patents

Abstract

The invention relates to pharmaceutical industry and medicine, in particular, to new PEG-interferon derivatives and to the discovery of a new functionally active, highly stable conjugate of interferon to polyethylene glycol with an activity of interferon alpha of general formulawhere n - a number of monomer chains in PEG with molecular mass from 20 to 40 kDa, excluding PEG with molecular mass 30 kDa; m - integer ≥4; IFN - natural or recombinant polypeptide having the activity of IFN-alpha. The invention also relates to drugs containing the declared conjugate of formula (I), to pharmaceutical compositions suitable for treatment of viral infections and cancer, as well as diseases associated with primary or secondary immunodeficiency, containing PEG-IFN conjugate of formula (I) and therapeutically acceptable excipients.

Description

The invention relates to pharmaceuticals and medicine, in particular, to new physiologically active interferon conjugates, in particular, to new interferon conjugates with polyethylene glycol, which can be used in medicine, for example, for the treatment of viral, immune and oncological diseases.

Interferons (IFN) are a group of biologically active proteins or glycoproteins produced by various cells in response to a viral infection or when certain chemical and biological substances are exposed to cells (Reika, 8., 1. Mar. 2007, 13; 282 (28 ), pp. 20047-20051). The binding of IFN to cellular receptors leads to the induction of the synthesis of a number of intracellular proteins that mediate the antiviral, immunomodulatory and antiproliferative effect of IFN (Reika, 8., e1 a1., 1tipo1od1ca1 Reu1e \\ b. 2004, 202, pp. 8-32; . 1., e1 a1., CGO 111 Ras1og8, 2004, 22 (4), pp. 243-251).

The genes of various human IFN were cloned and expressed in bacterial and animal cells; as a result, many recombinant IFNs were obtained and purified (Reika, 8., 1. Βίο1. Sc. 2007, 13; 282 (28), pp. 20047-20051) . Medicines containing recombinant IFNs are used in clinical practice for the treatment of a number of viral, oncological and immune diseases (Сеуайех 8., Pa \\ '1o1y <1.M., 2009, Napb. Exp. PagtasoG, (189), p. 203-41).

Currently, IFN-based drugs are used in clinical practice for the treatment of a number of viral, oncological and immune diseases. IFN drugs were most widely used for the treatment of viral hepatitis, which is one of the most serious social problems. At present, there are about 350 million chronically ill hepatitis B patients and 170 million hepatitis C patients in the world (R. MagseSp., Lepus lupus pylori, 2009, 29 (§1), pp. 1-8). In most cases, hepatitis C disease acquires a chronic course with the formation of severe outcomes — liver cirrhosis and hepatocellular carcinoma (Mob1 A.A. e1 a1., Oga1. Όίδ. 2008, 14 (1), p. 10-4). According to the WHO, since 1961, chronic hepatitis and cirrhosis of the liver, as a cause of death, have moved from 10th to 5th place in the USA and Western Europe (MagseSpn R., Briguet lneprina1, 2009, 29 (§1), pp. 1-8 ) The use of preparations containing IFN-α is also known for the treatment of a number of leukemia and solid tumors, including recurrent melanoma (Vikotek1 V. e1 a1., S1Sh. Opso1., 2002; 20 (18), pp. 3841-3849; Eesa1p5 M. e1 a1., VueGuide 2002; 16 (4), pp. 261-81; Oshp1a5-S.'agbata A. e1 a1., 8tt Tggot Netoy, 2006; 32 (4 P1 2), pp. 409-16 )

The efficacy of native IFN preparations is limited by rapid absorption from subcutaneous tissues, large distribution volume, relatively low stability, short half-life, high immunogenicity and toxicity (\ νί115 V.G., S1Sh Pbagtasoks. 1990; 19, p. 390-399.). As a result, within a few hours after administration, a rapid decrease in the concentration of IFN in the blood plasma is observed, while interferon is not detected in the plasma 24 hours after the injection (CbA1e1i1 E. e1 a1., Bg. 1. CI. Pbactaco. 1999; 47; , p. 365-371). A decrease in the concentration of interferon creates the conditions for the resumption of virus replication and an increase in the concentration of viral particles (Lamb / R e1 a1., E1d. Δδ. 8cg 1997: 42 (1), pp. 178-185). Therefore, there is a need for frequent injections of the drug to achieve effective therapeutic plasma concentrations, which leads to dose-related side effects. Due to these features, monotherapy of chronic hepatitis C (CHC) IFN-a2b for 12 months. led to a stable virologic response in only 15–20% of patients; in patients with genotype 1c and high virological load, the drug was not observed at all (MSNhcNiop 1. e1 a1., Ν. Eps1. 1. Meb. 1998: 339, 21, p. . 1485-1492).

The therapeutic efficacy of IFN can be increased by using prolonged-release drugs, in particular pegylated IFN (Mappk M.R. e1 a1., Lapse! 2001, 358, pp. 958-65; / echet 8. e1 a1., Exhre Ort 1puyev Aegidk 2001, 10 (12), pp. 2201-2213).

The preparation of PEGylated IFN consists in the chemical binding of an interferon molecule to a polymer - monomethoxypolyethylene glycol (mPEG), consisting of repeating subunits of ethylene oxide with a methoxy group at one end and a hydroxyl group at the other. The mPEG molecule may have a different molecular weight and stereochemical structure (linear or branched). To carry out the PEGylation reaction, the hydroxyl group at the end of the MPEG is activated by various reactive functional groups. An activated MPEG can covalently bind to a protein in one or several places, which depends on the nature of the activated group and the reaction conditions (2aHp§ku 8., Vusopf Szet. 1995: 6, pp. 150-165; Voeik ML., E1 a1. , Α6ν Egad Eeyu Ksu. 2002, 54 (4), pp. 459-476).

PEGylation of IFN leads to an improvement in pharmacokinetics, an increase in the half-life of blood, a decrease in blood concentration fluctuations, a decrease in immunogenicity and toxicity, an increase in the activity of w1uo (with a decrease in the activity of wuigo); increased stability (Ollie R. e1 a1., C11p. Pagtaco. Th. 2000, 68, pp. 556-67; Webbu K.V. e1 a1, Heploduod, 2001, 33, pp. 433-438).

The decrease in activity in the system is directly dependent on the molecular weight of the PEG used. The addition of small PEG molecules with a molecular weight of <5 kDa causes minor

- 1,020,257 changes in the activity of ίη νίΐτο (Stas, Μ. Ί .. s1 a1, ί. ΒίοΙ. Sit. 2005, 280, p. 6327-6336). The pharmacodynamic and pharmacokinetic properties of PEGylated proteins containing PEG with such a mass, and unmodified proteins are practically the same. The addition of PEG with a higher molecular weight and multiple sites leads to a significant decrease in the bioactivity of ίη νίΐτο, as a result of ineffective binding to the receptor, but in this case there is an improvement in the pharmacokinetic and pharmacodynamic properties of the PEGylated proteins (E1dabo C., e1 a1., Cg. Isu. Tyue. . Etade Sateg 8uz1 1992, 9, p. 249-304), which as a result leads to an increase in the biological activity of ίη ν Вνο (BaLop R., VePyo1b A., Ryagt., 8c1.Tesipo1. Tobau 1998, 1, p. 352- 356). The form of PEG also affects bioactivity and pharmacokinetic properties - the conjugate with linear PEG extends to a larger volume than with PEG with a branched structure (Sayseb R., Uegopehe Ρ.Μ, Α6ν. Etad. Eey. 'Isu. 2003; 55 ( 10), pp. 1261-1277).

The antiviral activity and biological properties of PEG-IFN conjugates also largely depend on the activated mPEGs used, the functional groups of which differ in their ability to modify various amino acid residues of the protein and in the type of chemical bonds formed with the protein (^ Leyk, E., e! A1. , Α6ν Etad Eey 'Isu. 2002, 54 (4), pp. 459-476). In the preparation of pegylated proteins, activated mPEGs that can bind to the free amino groups of proteins (succinimidyl carbonate, succinimidyl succinate, trasylate, triazine, hydroxysuccinimide esters, acetaldehyde, etc.) found the widest application.

Various PEG-IFN conjugates are known in the art.

Known conjugate PEG-IFN-a2a with linear PEG with a molecular weight of 1-5000 Da (Patent I. 8. Νο. 5382657 (1995)). For conjugation, ethanediol derivatives of MPEG were used; the reaction was carried out at pH 10 at room temperature for 0.5–4 h. A 3-fold excess of PEG was used to carry out the PEGylation of IFN. Under the conditions used, the binding of PEG to IFN occurred through the sterically accessible free amino groups of various lysines (Lb8-31, Lb8133, Lb8134, Lb823, Lb8131, Lb 121, Lb870, Lb583, Lb849 and Lb8112) with the formation of carbamide bond 8. ! a1., Αηа1уΐ ^ са1 ВюсюетЩту 2007, 247, p. 434-440). Thus, the obtained PEG-IFN-a2a conjugate consisted of a mixture of positional isomers, where each isomer contained one PEG. In comparison with unmodified IFN, the specific antiviral activity of the isomers ranged from 6% (for Ly8112) to 40% (for Ly8133). The total specific activity of the obtained conjugate, consisting of 11 isomers, was 34% of unmodified IFN. The activity of the obtained ίη νίΐτο conjugates also depended on the molecular weight of PEG and varied from 45% (for PEG - 25 kDa) to 25% (in the case of PEG - 10 kDa). The disadvantages of the resulting conjugate are:

1. The use of ethanediol-activated MPEG with a low molecular weight (<5 kDa), as a result of which the pharmacokinetic parameters of the obtained PEG-IFN conjugate and unmodified IFN did not differ significantly, and therefore this conjugate was not used in the clinic.

2. The formation of a large number of positional isomers with different specific activity, in which PEG was bound to the IFN molecule via free amino groups of various lysines.

Known conjugates of PEG-IFN-a2b obtained by conjugation of a native IFN-a2B with linear (patent VI 2311930 (2004)) or branched (patent KP 2382048 (2008)) mPEG derivatives with a molecular weight of 13-17 kDa. The reaction was carried out at a pH of 9.5 for 60 h (for linear mPEG) or at a pH of 7.5 for 12 h (for branched mPEG) using a 50-100-fold molar excess of activated PEG. As a result, PEG-IFN conjugates were obtained, the total antiviral activity of which varied from 29 to 38% of the activity of unmodified IFN. Data on the stability of the bond, the number of positional isomers and their specific antiviral activity are not given. The disadvantages of the obtained conjugates are:

1. The use of trisyl derivatives of activated MPEG with a half-life in aqueous solutions of less than 20 minutes Previously, it was shown that, in the interaction of trisylate derivatives of PEG with proteins, in addition to stable amide bonds through amino groups, unstable sulfamate bonds are also formed (Sab & Kirrei, 1995, Teΐ ^ ayeb ^ οη 1eyet8, 22 (36), p. 3837-3838).

2. The binding of trisyl-activated PEG to proteins occurs through all sterically accessible free amino groups of lysine (^ Leyk e! A1., 2002, Α6ν Aegid Eey 'Isu., 54 (4), pp. 459-476), which leads several positional isomers.

3. The PEGylation of IFN at high pH (pH 10) for a long time (60 h) can lead to a change in the structure of IFN.

4. A significant excess of activated PEG during the PEGylation reaction of IFN (molar ratio of PEG / protein was 50-100 / 1) leads to a high cost of the obtained product.

Known conjugate PEG-IFN-a2b obtained by binding of native IFN-a2B with a branched triazine derivative of MPEG with a molecular weight of 7.5-35 kDa (patent KI 2298560, 2004), the total antiviral activity of which amounted to 6.4% of the activity of unmodified IFN . Data on the stability of communication, the number of positional isomers and their specific antiviral

- 2 020257 activity not shown.

The disadvantages of the resulting conjugate are:

1. The use of triazine chloride derivatives of activated MPEG, which, in addition to free amino groups, can bind to functional groups of other amino acids - serine, tyrosine, threonine and histidine, which leads to the appearance of many isomers, some of which are characterized by an unstable bond. Moreover, triazine derivatives are not currently used due to their high toxicity (Uegopeve & Ravi !, Aegid Όίβεον. Tobau, 2005, 1: 10 (21), p. 14511458).

2. Low specific activity of the conjugate PEG-IFN.

Known conjugate PEG-IFN-a2a obtained by conjugation of IFN-a2a with branched PEG with a molecular weight of 40 kDa (patent KI 2180595, 1997). For conjugation, a идhydroxysuccinimide ester derivative of MPEG was used, selectively interacting with available free amino groups of the protein to form a stable amide bond. The reaction was carried out at pH 9 at 4 ° C for 2 h with a mPEG / protein ratio of three. The reaction was stopped and the resulting conjugate was purified on a sorbent Bgas1ode1 М CM 650 (M). The yield of purified conjugate was 40-45%. In this case, the IFN-PEG conjugate was obtained, consisting of 6 positional isomers, each of which was linked to a single PEG molecule via a stable bond through the free amino groups of lysines - Buv31, Buv121, Bui 131, Bui 134, Bui 70 and Bui 83 (Vabop R ., e! a1., Vyusonshch. Sesh. 2001; 12, p. 195-202; Boveg B, e! a1., Prgoes eshr. RipG. 2003, 30 (1), pp. 78-87). The isomers associated with PEG, through lysines at position 31, 121, 131, and 134, totaled 94%. The activity of the total conjugate, consisting of 6 positional isomers, amounted to 1-7% of the native IFN-a2a (Voi1evbp A., e! A1., PrgieGsgop & Sucker Kee. 2006, 26, p. 849-853). Despite the low antiviral activity of w νί6Ό, this conjugate, in comparison with unmodified IFN, had improved pharmacokinetic properties (Bima Μ., E! A1., 1oigpa1 Nera! Oduod 2006, 45, p. 204-213), and is currently used in a clinic called Pegasis (manufactured by Hoffmann-La Roche).

The disadvantages of the resulting conjugate are:

1. The use of activated PEG Ν-hydroxysuccinimide ester, which binds to all sterically accessible amino groups; as a result, the resulting conjugate was a mixture of 6 isomers differing in specific activity.

2. Low antiviral specific activity of the obtained conjugate w x'Pgo.

The prototype for the present invention is the conjugate PEG-IFN-a2b described in patent IB 5951974 (1999). For the PEGylation reaction, linear MPEG activated by succinimidyl carbonate group (BS-shRES) with a molecular weight of 5 to 12 kDa was used. According to the described method, a conjugate of IFN with PEG with a molecular weight of 12 kDa was obtained, which is currently used in the clinic for the treatment of viral hepatitis - the drug Pegintron (company B11eppd-P1oid11. USA).

It was established that the obtained PEG-IFN-a2b conjugate (Pegintron preparation) consists of 13 positional isomers, each of which contains one PEG molecule, attached to different parts of the protein molecule (^ apt, Υ.-B. e! A1., Vusyesh18bu, 2000, 39 (35), pp. 10634-10640; Sgase Μ., E! A1.,

I. 1n! Er £ egop Su! Okay Kee. 2001, 21, pp. 1103-1115; Ипοипдβ! E ^ B., e! A1., Sigg Ragsh Oev. 2002; 8 (24), pp. 2139-2157). It was shown that the PEG molecule was bound to interferon not only through the free amino groups of lysines (Bouv 31 Bouv 49, Bouv 83, Bouv 121, Bouv 131, Bouv 133, Bouv 134 and Bouv 164) and Ν-terminal cysteine (Suv1), but also through the imidazole ring of histidine (H1v7, H1v34), tyrosine (Tug129) and the OH group of serine (Beg163). The content of individual isomers ranged from 0.8 (Tug129) to 47% (H1v34). The antiviral activity of the total Pegintron preparation, consisting of 13 positional isomers, amounted to 28% of the native protein. The specific activity of the individual isomers varied from 11 to 37% of the native IFN-a2b (Woopdv1eg B., e! A1., Sigg Pbt Oev. 2002; 8 (24), pp. 2139-2157). The highest specific activity was in the main isomer conjugated to PEG via H1b34, which amounted to 37% of the native protein. The free amino groups of IFN were coupled to PEG via a stable bond, while in the main isomer, approximately 47%, PEG was linked to the imidazole ring of histidine (H1b34) via an unstable (in aqueous solutions) carbamate bond (Coebb Μ.Ε, e! A1 ., Α6ν Bgad BeRm Kem. 2002, 54 (4), pp. 459-476).

The pharmacokinetic parameters of the resulting conjugate were better than those of unmodified IFN (Bbma, E., e! A1., 1o1 Nera1odu 2006, 45, p. 204-213).

The disadvantages of the resulting conjugate are:

1. The use of succinimidyl carbonate activated PEG binding not only to the free amino groups of IFN, but also to the functional groups of other amino acids. As a result, the resulting conjugate consisted of a mixture of 13 positional isomers differing in antiviral specific activity.

2. The formation of an unstable imidazole-carbonate (carbamate) bond, mainly (Н1в-34) iso

- 3,020,257 least of the PEG-IFN conjugate leads to the fact that when the drug Pegintron is ingested, the native IFN is hydrolyzed and released, which has a biological effect. Thus, the Pegintron preparation is best regarded as a pro-drug, which, after introduction into the body, breaks down with the formation of active interferon (Uoyidieg 8., C1 A1., Sigg Ryatt Eek. 2002; 8 (24), pp. 2139-2157). Due to the instability of PEG-IFN in solution, the dosage form of Pegintron is stored only in the form of a freeze-dried powder.

The objective of the present invention was to obtain a new stable conjugate of pegylated IFN, with the activity of IFN alpha, consisting of a single positional isomer, with improved stability, with reduced immunogenicity, with improved pharmacokinetic parameters, with an optimal combination of molecular weight parameters of PEG and antiviral activity suitable for creating medicinal product for medical purposes, contributing to the expansion of the arsenal of drugs of a given focus, pharmaceutics eskoy composition based on the resulting PEG-IFN.

The problem is solved by creating a new molecule of a functionally active PEG-IFN conjugate with interferon-alpha activity, in which a linear PEG molecule with a molecular weight of 20 to 40 kDa, with the exception of PEG with 30 kDa, is connected to the IFN-α molecule by a stable bond strictly through the alpha amino group The Ν-terminal cysteine so that the general formula of the PEG-IFN conjugate with linear mPEG is the following compound (I):

O) where η is the number of monomer units in a PEG with a molecular weight of 20 kDa to 40 kDa, with the exception of PEG with a molecular weight of 30 kDa;

t is an integer> 4;

ΙΡΝ - a natural or recombinant polypeptide having IFN-alpha activity.

In the conjugate obtained, a linear PEG with a molecular weight of 20-40 kDa, with the exception of a PEG of a molecular weight of 30 kDa, is associated with the alpha-amino group of the кон-terminal amino acid of IFN.

The selectivity of IFN modification strictly according to the alpha-amino group of the кон-terminal amino acid is achieved through the use of aldehyde-activated mPEGs of the general formula (II) and the PEGylation reaction at pH <6:

(Ii)

where η is the number of monomer units in a PEG with a molecular weight of 20 to 40 kDa, with the exception of PEG with a molecular weight of 30 kDa;

t is an integer> 4;

The optimal ratio of the parameters of the molecular weight of PEG and antiviral activity is achieved through the use of activated MPEG (II) with a value of m> 4.

The decrease in immunogenicity, toxicity and improvement of pharmacokinetic parameters is achieved by increasing the molecular weight of the attached PEG. New in comparison with the prototype is:

1. To obtain the conjugate PEG-IFN used aldehyde derivatives of activated mPEG.

2. The structural formula of the conjugate PEG-IFN.

3. The resulting PEG-IFN conjugate is represented by one positional isomer.

4. A stable bond between the PEG molecule and the IFN molecule.

5. An increase in the molecular weight of the PEG-IFN conjugate due to the size of the attached PEG.

6. Improved pharmacokinetic parameters.

To obtain the inventive PEG-IFN conjugate, recombinant human IFN-a2b (manufactured by Biocad CJSC) and butyr or pentalaldehyde mPEG derivatives of formula (II) with a molecular weight of 20 kDa were used.

The conjugation reaction was carried out at a pH below 6.0 in the presence of a reducing agent at a temperature equal to or below 20 ° C. The PEG / protein molar ratio was 2.5-5 / 1. PEG-IFN conjugate formation was monitored by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (DDS) under reducing conditions and reverse phase high performance chromatography (RP-HPLC). The purification of mono-PEG-IFN from the reaction products of unmodified IFN and from undesirable forms of PEG-IFN containing two or more PEG molecules per protein molecule was carried out by chromatography on cation-exchange sorbents. The elution of mono-PEG-IFN was carried out with a concentration gradient of sodium chloride from 0.05 to 0.2 M in

- 4 020257 ferric solution with a pH below 6. The purified mono-PEG-IFN preparation was dialyzed against a 10-50 mM buffer solution with a pH of 4-5, salts or polysaccharides, or polyhydric alcohols, or polyvinylpyrrolidones, or monosaccharides, or amino sugar, or proteins were added. , or amino acids and non-ionic detergents and stored in plastic or glass bottles with a siliconized surface at a temperature of 4 ± 2 ° C.

To characterize the obtained monoPEG-IFN-a2b conjugate, its purity, homogeneity, physicochemical, biological and pharmacokinetic parameters were studied in comparison with unmodified IFN.

The present invention is illustrated by figures of a graphic image, which presents:

FIG. 1 - kinetics of the formation of the conjugate of IFN-a2b during the reaction using the butyraldehyde derivative of MPEG;

FIG. 2 - reverse phase HPLC of the PEG-IFN-a2b conjugate on a column of 8outte! C18 (4.6 x 150 mm).

FIG. 3 - electrophoretic analysis of the conjugate PEG-IFN-a2b in comparison with the modified IFN-a2b;

FIG. 4 - MABO1 mass spectra of modified IFN-a2b and PEG-IFN-a2b conjugate;

FIG. 5 — localization of the PEGylation site in the PEG-IFN-a2b conjugate. Comparison of the mass spectra of the tryptic peptides of unmodified IFN-a2b (A) and the PEG-rchIFIF-a2 (B) conjugate in the t / ζ range 1200-1400;

FIG. 6 - thermal stability of the conjugate PEG-IFN-a2;

FIG. 7 - proteolytic stability of the conjugate PEG-IFN-a2b when treated with trypsin;

FIG. 8 - immunogenicity of the conjugate PEG-IFN-a2b in comparison with unmodified IFN-a2b;

FIG. 9 - pharmacokinetics of the conjugate PEG-IFN-a2b in comparison with unmodified IFNa2b.

Examples of specific performance of the method for producing PEG-IFN-a2b and studies of its properties are given below.

Conjugates of PEG-IFN-a2 of formula (I), which are the object of the present invention, can be used to obtain drugs for the prevention and / or treatment of viral diseases, because in addition to high stability and reduced immunogenicity, they have a high antiviral activity. Also, pharmaceutical compositions containing as an active ingredient an effective amount of a conjugate of the formula (I) and obtained by known methods used in pharmaceuticals can be used both individually and in combination with other therapeutic agents (e.g., ribavirin) for the prevention and / or treatment of viral diseases (chronic active hepatitis (in particular, hepatitis B and C)) (1au N. e! a1., Thyete Eid1aib 1. ίί tebuche 2006, 356, pp. 1269-1271; MsNi1sy8oi 1. e! a1., THe No. \ ν Eid1aib 1. οί Tebuche 2009, 361, pp. 580-593, 2009). The object of the present invention is also a method for the prevention and / or treatment of viral diseases, for example hepatitis C and hepatitis B, comprising administering a therapeutically effective amount of the inventive conjugate of formula (I).

It is also known from the prior art that IFN-α and PEGylated interferons-a are used as immunomodulators for the treatment of oncological diseases, in particular, hairy cell leukemia, laryngeal papillomatosis, melanoma, renal carcinoma, myeloid leukemia, Kaposi’s sarcoma (Esa1p8 M., e! A1x, Vue ; 16 (4), pp. 261-281; Vikotekt V., e! A1., SI. Oiso1. 2002; 20 (18), p. 38413849; OshShch5-Sagbata A. e! A1, 8sht Tyto Netoz !. 2006; 32 (4 P! 2), pp.: 409-16; Lodia1 e! A1., Eig 1 Egta1o1. 2008 1ai-Reb; 18 (1), pp. 29-35; Caeet e! A1, Eig 1 Saiseg. 2010 1ai; 46 (1), pp. 41-46). Based on the known etiopathogenesis of these diseases and the use of IFN-α, as well as conjugates for their prophylaxis and / or treatment, the objects of the present invention are also drugs and pharmaceutical compositions containing the claimed PEG-IFNα conjugate in an effective amount, having antiproliferative and immunomodulatory effects that can be used for the prevention and / or treatment of cancer and diseases accompanied by primary or secondary immunode deficiency conditions, and also an object of the present invention is a method for the prevention and / or treatment of cancer and diseases accompanied by primary or secondary immunodeficiency conditions, comprising administering a therapeutically effective amount of a PEG-IFN-α conjugate of formula (I).

A conjugate of formula (I) with an optional pharmacologically acceptable excipient, diluent and / or pharmaceutically acceptable excipients is administered intravenously, subcutaneously, intramuscularly or in any other suitable way, for example, in the form of capsules, syrup, spray, drops, injections or suppository. The route of administration varies depending, for example, on symptoms and age, the frequency of administration and the interval between administrations vary depending on the disease and its severity or on the purpose (therapeutic or prophylactic use), effective

- 5,020,257 the amount of active principle of PEG-IFN-α is selected taking into account the above factors.

As pharmaceutically acceptable auxiliary components that can be included in the pharmaceutical composition with PEG-IFN-a2, buffer salts (e.g., acetate, citrate, hydrogen-carbonate, phosphate buffers), stabilizers (e.g., polysorbates, EDTA, polyvinylpyrrolidones can be used) , dextrans, HSA, paraoxybenzoic acid esters, alcohols (e.g. benzyl alcohol), phenols, sorbic acid), a disinfecting component (e.g. benzyl alcohol), an osmotic pressure regulator (e.g. sodium chloride, chl potassium chloride, polyols, e.g. glycerin, arabitol, sorbitol, mannitol, lactose, dextran), surfactants (e.g. HSA, polyvinylpyrrolidone, lecithin, tween 80, polyoxyethylene-polyoxypropylene copolymer, casein), surfactants (e.g. block copolymers of ethylene oxide and propylene oxide, propylene oxide and ethylene oxide, sorbitan monolaurate, sorbitol ester, polyglycyrin fatty acid ester, cocamide DEA lauryl sulfate, alkanolamide, stearite polyoxyethylene propylene glycol, polyoxyethylene lauric ester, cetyl polyoxyethylene, polysorbate, glycerol monostearate, glycerol distearate, sorbitol monopalmitate, polyoxyethylene sorbitan monooleate and polyoxyethylene sorbitan monolaurate and propylene glycol monostearate), antioxidants (for example, Trilon B, bysteine, sodium sulfite, acrylhydrochloride, ascorbate sodium cyanide aminoglide, ascorbide sodium dorbate acid) and diluents (e.g. water).

Example 1. Obtaining the conjugate PEG-IFN-a2 using a butyraldehyde derivative mPEG.

To 785 ml of a buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.0) containing recombinant IFN-a2 at a concentration of 1.7 mg / ml, 16 ml of a 1M sodium cyanoborohydride solution was added, mixed and 5 g was added. dry butyraldehyde derivative of PEG with a molecular weight of 20,000 Da. The mixture was thoroughly mixed and incubated for 22 h at a temperature of 17 ± 3 ° C. At various time intervals, 50 μl aliquots were taken from the reaction mixture and the kinetics of the formation of the PEG-IFN-a2 conjugate were analyzed using the SDS page electrophoresis method in the presence of DDS. For this, 1/3 of the buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% DDS, 0.005% bromphenol blue was added to the selected sample and heated for 3 min in a boiling water bath. Samples in a volume of 5 μl were introduced into the wells of the prepared PAAG plates and electrophoresis was performed in 12.5% PAAG in the presence of DDS. At the end of electrophoresis, the gel was stained with Coomassie B-250. The kinetics of the formation of the PEG-IFN-a2 conjugate is shown in FIG. 1. At the point in time when the content of PEG-IFN was more than 70%, the reaction mixture was diluted 10 times with 5 mm sodium acetate buffer, pH 5.0.

Example 2. Obtaining the conjugate PEG-IFN-A2 using pentalaldehyde derivative mPEG.

To 100 ml of a buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5), containing recombinant IFN-a2b at a concentration of 2.2 mg / ml, 2.05 ml of a 1M sodium cyanoborohydride solution was added, mixed and 0.9 g of dry pentaldehyde derivative of PEG with a molecular weight of 20 kDa was added. The mixture was thoroughly mixed and incubated for 24 hours at a temperature of 6 ± 2 ° C. At different time intervals, 50 μl aliquots were taken from the reaction mixture and the kinetics of the formation of the PEG-IFN conjugate was analyzed using the PAGE method as described in Example 1. At the time when the PEG-IFN content was more than 70%, the reaction mixture was diluted 10 times 5 mM sodium acetate buffer, pH 5.0.

Example 3. Purification of the conjugate of mono-PEG-IFN-A2 using ion-exchange chromatography on a cation-exchange sorbent.

The diluted solution obtained in Example 1 was applied to a column with a cation exchange sorbent (CM-Sepharose, 300 ml) balanced with 5 mM sodium acetate buffer, pH 5.0, (buffer A) at a rate of 5 ml / min. The sorbent column was washed sequentially with buffer A and buffer A containing an NaCl concentration gradient from 0.05 to 0.2 M. Aliquots were taken from each fraction for analysis of the samples by electrophoresis in PAG as described in Example 1. Fractions containing the mono-PEGylated PEG-conjugate IFN-a2 was combined, dialyzed against 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, sterile filtration was performed and stored at 4 ± 2 ° C.

Example 4. Purification of the conjugate monoPEG-IFN-a2b using ion exchange chromatography on a cation exchange sorbent.

The diluted solution obtained in Example 2 was applied to a column with a cation exchange sorbent (BR-Sepharose, 50 ml) balanced with 5 mM sodium acetate buffer, pH 5.0, (buffer A) at a rate of 5 ml / min. The sorbent column was washed sequentially with buffer A and buffer A containing a concentration gradient of 1C1 from 0.03 to 0.3 M. Aliquots were taken from each fraction for analysis of the samples by electrophoresis in SDS page as described in Example 1. Fractions containing mono-PEGylated conjugate PEG-IFN-a2 was combined, dialyzed against 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, sterile filtration was performed and stored

- 6,020,257 at a temperature of 4 ± 2 ° C.

Example 5. Analysis of the conjugate PEG-IFN-A2 using the method of reverse phase high performance liquid chromatography.

The PEG-IFN-a2 conjugate obtained according to claim 3 was diluted with 20 mM sodium acetate buffer with a pH of 5.0 to a concentration of 0.1 mg / ml, and 100 μl of the obtained sample was added to an 8 out column of C18 (4.6 x 150 mm). The study was carried out on a Wgehech chromatograph of the company \ ya) ex5 at 214 nm. From the results presented in FIG. 2, it follows that according to RP HPLC, the purity of the claimed PEG-IFNα2 conjugate is more than 99%.

Example 6. Determination of the level of endotoxins.

The content of bacterial endotoxins (BE) in the samples of the PEG-IFN-a2 conjugate prepared according to claims 3 and 4 was determined using ίΐη νίΐΓΟ using the LAL test (gel-thrombus test modification) in accordance with the requirements of the General Pharmacopoeia Monograph 42-0002-00. We used a diagnostic multicomponent kit of the company L55OS1a1e5 o £ CARE ESR. 1ps LAL reagent RUVOTEЬ with a sensitivity of 0.03 EU / ml, control endotoxin standard (KSE, 0.5 μg in a vial), water for the LAL test. From the results presented in table. 1, it can be seen that the content of BE in the conjugate samples is less than 1.5 units of endotoxin (eE) per mg of protein, which is much lower than the level of BE allowed for medications based on recombinant proteins.

Table 1. The content of bacterial endotoxins in the conjugate PEG-IFN-a2

The drug conjugate PEG-IFN-a2 The content of endotoxins (eE / mg protein) PEG-IFN-a2 obtained in example 3 <1.5 PEG-IFN-a2 obtained according to example 4 <1.4

Example 7. Electrophoretic analysis of the conjugate PEG-IFN-a2b in comparison with unmodified IFN-a2b.

A sample of the conjugate PEG-IFN obtained in example 3 was analyzed by electrophoresis in SDS page, as described in paragraph 1. Electrophoresis was performed with a load of 40 μg of protein per well under non-reducing conditions. Proteins in the gel were stained with dye.

Coomassie B-250. At the same time, electrophoresis of unmodified IFN-a2 was performed. The PEG-IFN conjugate was represented by a single band (Fig. 3, track 1). From the electrophoregrams of the PEG-IFNα2 conjugate and unmodified IFN-a2 shown in FIG. 3, it can be seen that the PEG-IFN-α conjugate has a higher molecular weight than rhIFN-α (Fig. 3, tracks 1 and 2). It should be noted that the exact molecular weight for PEGylated proteins cannot be determined by the EF method, since the addition of a hydrophilic PEG molecule to a protein significantly increases the Stokes radius of the complex formed. As a result, the progress of the PEG-protein complex in the gel slows down, and its molecular weight is much higher than the sum of the molecular weights of the protein and PEG.

Example 8. Determination of the molecular weight of the conjugate PEG-IFN-a2 in comparison with unmodified IFN-a2 by mass spectrometry.

μl of a sample of the PEG-IFN conjugate obtained according to claim 3, and 0.3 μl of a solution of 2,5-dihydroxybenzoic acid (A1bps11, 10 mgml ^-1 in 20% acetonitrile in water with 0.5% TFA) were mixed and dried in air . In a similar way, a sample of unmodified IFN-a2 was prepared.

Mass spectra were obtained on an Ingayeh II VVIKEV) time-of-flight mass spectrometer (Germany) equipped with a UV laser (N6). Mass spectra were obtained in the mode of positive ions, in the linear mode, the error in measuring the average mass does not exceed 10-15 Da.

The results of mass spectrometric analysis of unmodified rhIFN-a2 and PEG-IFN-a2 conjugate in the t / ζ range from 10000-50000 are shown in FIG. 4. From FIG. 4A, the rhIFN-a2 mass spectrum contains the main peak corresponding to the singly charged [M] ⁺ ion with a molecular weight of 19.295 kDa.

In the mass spectrum of the PEG-IFN-a2 conjugate shown in FIG. 3B, a diffuse main peak is shown corresponding to a singly charged [M] ⁺ ion with a molecular weight of 40.498 kDa. Peak blur is associated with the heterogeneity of monomethoxyPEG preparations. For the PEG-IFN conjugate, the measured t / ζ value at the peak maximum (40.498 kDa) coincides with the calculated data on the sum of the molecular weights of IFN-a2 (19.295 kDa) and the attached PEG (20 kDa).

Example 9. Localization of the site of PEGylation in the conjugate PEG-IFN-a2b.

To 5 μl of a sample of the PEG-IFN conjugate obtained in Example 3, 5 μl of a solution of modified trypsin (Rgoted) in 0.05 M XN ₄ HCO ₃ with a concentration of 15 μgml ^{-1 was} added. The hydrolysis was carried out for 16 h at 37 ° C, then 10 μl of 0.5% TFA in a 10% solution of acetonitrile in water was added to the solution and thoroughly mixed. In a similar way, a sample of unmodified IFN was prepared. The resulting solutions were used to obtain ΜΑΕΌΙ-mass spectra.

The localization of the PEG binding site with the IFN molecule was determined by comparing the mass spectra

- 7 020257 tryptic hydrolyzate of the conjugate PEG-IFN-a2 and unmodified rhIFN-a2. In the tryptic hydrolyzate of the PEG-IFN-a2 conjugate, there should be no peptide corresponding to the portion of the protein through which the modification occurred. In the table. 2 shows the mass spectra of experimental tryptic peptides for unmodified IFN-α and PEG-IFN-a2 conjugate. The table shows that the mass spectra of the tryptic peptides of unmodified IFN-a2b almost coincide with the mass spectra of the tryptic peptides of the PEG-IFN-a2 conjugate (Table 2), but there is no peptide with a molecular weight of 1313.649 in the tryptic digest of the PEG-IFN-a2 conjugate present in the tryptic digest of unmodified rhIFN-a2 (see also Fig. 5). According to the analysis of the theoretical masses of the tryptic hydrolyzate of IFN-a2, the peak with t / ζ 1313.649 corresponds to the Ν-terminal protein peptide (Table 2). Its absence in the tryptic digest of the PEG-IFN-a2 conjugate indicates a modification of the кон-terminal peptide, which, having become heavier by the mass of PEG, is outside the studied region of the spectrum. The binding of PEG to the IFN molecule occurred at the only free amino group present in this peptide - at the amino group of the Ν-terminal cysteine.

Table 2. Experimental mass spectra of tryptic peptides of the conjugate PEG-IFN-a2 and unmodified IFN-a2 in comparison with theoretical values

Experimentally detected peptides [M + TG Theoretical peptides IFN-a2b PEG-IFN conjugate Weight [M + NT Peptide Sequence 1313.649 - 1313.6266 1-12 SOURZSTN8O8K 1232.720 1232.673 1232.6966 13-22 KTM'AOMK. 1076.598 1076.558 1076.5955 14-22 TWOAOMK 910.463 910.463 910.5066 24-31 18Е8СЬК 2225.976 2225.928 2225.9999 32-49 ΏΚΗϋΡΟΡΡΟΕΕΡΟΝΟ 2459.264 2459.239 2459.3002 50-70 AET1RUNEMK) TSPT4 772.447 772,421 772.456 99-106 νιρονοντ 902.471 902.443 902.4941 113-120 EOZSHAUK 1030.590 1030.546 1030.5891 113-121 ЕО81ЬАУКК 741.431 741.424 741.4042 121-125 KUROK 750.492 750.476 750.4760 126-131 GGUK 1337.695 1337.651 1337.6670 134-144 KU8RSA) UEUUK 1209.565 1209.539 1209.5721 135-144 URSAAUEUUUK 1481.783 1481.735 1481.7594 150-162 8Е8Ь8ТЫЕЕЬЬК

Example 10. Determination of the specific antiviral activity of the conjugate PEG-IFN-a2b.

In the samples obtained according to example 3 and example 4, the antiviral specific activity was studied according to the protocol described in the European Pharmacopoeia using a culture of transplantable cells ΜΒΌΚ sensitive to alpha-type interferon and vesicular stomatitis virus (U8U). In the table. 3 presents the results of a study of antiviral activity. The international reference standard was used as the standard. From the table. Figure 3 shows that, despite the addition of a PEG molecule with a molecular mass of 20 kDa, the antiviral activity of the resulting conjugates is 42-45% of the activity of unmodified IFN-a2. It should be noted that the PEG-IFN conjugate described in the prototype method containing PEG with a molecular weight of 12 kDa had a lower activity (28-30%).

Table 3. Specific antiviral activity of the PEG-IFN-A2 conjugate compared to unmodified IFN-A2

A drug Specific antiviral activity (IU / mg) % of the activity of unmodified IFN-a2b IFN-a2 2.1 x 10 ⁸ one hundred% The conjugate PEG-IFN-A2, obtained according to p.Z. 0.7 x 10 * 40% The conjugate PEG-IFN-a2 obtained according to claim 4. 0.9 x 10 * 45%

Example 11. The study of thermal stability of the conjugate PEG-IFN-a2b.

ml of a sample of the PEG-IFN conjugate obtained in Example 3 was placed in a water bath at a temperature of (50 ± 2) ° С and the appearance of turbidity in the protein solution was examined at various time intervals by measuring the optical density at 340 nm. A similar study was conducted of the thermal stability of unmodified IFN-a2. The formation of insoluble protein aggregates led to the appearance of turbidity and to an increase in optical density at 340 nm. In FIG. 6 presents the results

- 8,020,257 studies. During the observation period (28 hours), the PEG-IFN-a2 conjugate remained stable, while unmodified IFN-a2b almost completely precipitated after 28 hours.

Thus, the data obtained allow us to conclude that a significant increase in the thermal stability of the PEG-IFN-a2 conjugate is compared with unmodified protein.

Example 12. The study of the proteolytic stability of the conjugate PEG-IFN-a2b when treated with trypsin.

To 1 ml of the PEG-IFN conjugate obtained in Example 3, 150 μl of 1M Tris-HCl buffer, pH 8.5, 2 μl of 0.5 M CaCl ₂ solution and 15 μl of trypsin (Rgoted) with a concentration of 20 μg / ml were added. Samples were incubated at 37 ° C. At certain time intervals, 100 μl aliquots were taken and 150 μl of a 0.5% trifluoroacetic acid solution was added to stop the reaction. Similarly, samples containing unmodified IFN-a2 were prepared. Next, the samples were analyzed using reverse phase HPLC and the area of the main peak was determined. The test results shown in FIG. 7 show that, when treated with trypsin, the proteolytic stability of the PEG-IFN-a2 conjugate is higher than that of unmodified IFN-a2B.

Example 13. Determination of the stability of the conjugate PEG-IFN-a2b during storage.

Aliquots were taken from the sample obtained in Example 3 into sterile tubes with Eppendorf caps. The protein concentration in the PEG-IFN-a2 conjugate was 1 mg / ml. Samples were placed in a refrigerator at a temperature of (6 ± 2) ° С. At certain intervals, samples were taken and the specific antiviral activity and homogeneity of the preparation were analyzed using RP HPLC and electrophoresis (EP) in PAGE in the presence of DDS under non-reducing conditions, as described in Example 1. From the data in Table 1. Figure 4 shows that when stored at a temperature of (6 ± 2) ° C, the PEG-IFN-a2 conjugate is stable for at least 24 months.

Table 4. The stability of the conjugate PEG-IFN-a2b at a temperature of (6 ± 2) ° C

Options Shelf life (months) 0 3 6 nine 12 eighteen 24 Activity (IU / ml) 0.7x10® 0.75x10® 0.7x10® 0.75x10® 0.72x10® 0.7x10® 0.75 xU® Homogeneity,% (RP HPLC) 99.67 99.28 99.61 99.30 99.6 99.21 99.35 Homogeneity,% (EF in PAG) Y98 th 98 > 98 > 98 > 98 > 98 > 98

Example 14. The study of the immunogenicity of the conjugate PEG-IFN-a2b.

The PEG-IFN conjugates obtained according to example 3 and example 4 were diluted to a concentration of 5 million IU / ml and 200 μl (1 million IU / mouse) were administered intramuscularly to 1CP mice with a body weight of 20-22 g once a week, in for 5 weeks (5 groups of 5 mice per group). In parallel, a sample of unmodified IFN was prepared and similarly administered to mice at a dose of 1 million IU. At the end of the fifth week, animals were bled using retroorbital puncture. Blood serum was obtained by the standard method. The titer of antibodies in sera obtained by immunizing mice with unmodified IFN and PEG-IFN conjugates was determined using the direct enzyme-linked immunosorbent assay (ELISA). For this, 100 μl of a solution of unmodified IFN-a2 at a concentration of 200 ng / 100 μl was added to the wells of the microplate. Antisera obtained from different groups of animals were introduced into the wells of the tablet in a series of serial dilutions twice. Anti-mouse immunoglobulins conjugated with peroxidase were used to detect the formed immune complex. The titer of antiserum after administration of unmodified IFN was 1/1024. The titer of antisera after administration of PEG-IFN conjugates was 1/128. The results of the study are presented in FIG. 8.

Thus, it follows from the obtained results that the immunogenicity of the obtained PEG-IFN-a2 conjugates is 8 times lower than that of unmodified IFN.

Example 15. The study of the pharmacokinetics of the conjugate PEG-IFN-a2b.

A sample of the PEG-IFN conjugate obtained in Example 3, at a dose of 1 million IU, was administered intraperitoneally to male mice. In parallel to another group of mice, unmodified IFN-a2 was administered. At certain intervals, blood was collected from animals using retroorbital puncture. Blood serum was obtained by the standard method. The presence of interferon in serum was determined by antiviral activity. The pharmacokinetic results are presented in FIG. 9. From the results it follows that the inventive conjugate PEG-IFN-a2 has a significant prolonged effect. With the introduction of native IFN-a2, its content in the blood of animals reached a maximum 1 h after administration and then decreased very rapidly. With the introduction of the conjugate PEG-IFN, the maximum content of IFN in the blood of animals was observed after 12 hours (Fig. 9), and this level was maintained for

- 9,020,257 hours, and only then did the slow decrease in the content of IFN-a2 occur over 350 hours

The area under the curve for the inventive PEG-IFN conjugate was more than 50 times higher than the corresponding value for unmodified IFN. It should be noted that for the prototype PEG-IFN-a2b conjugate described in the method associated with PEG with a molecular weight of 12 kDa, the area under the curve exceeded the area under the curve for IFN by only 3-10 times.

Based on the results obtained (Fig. 9), the basic pharmacokinetic parameters for the inventive PEG-IFN-a2 conjugate were calculated. The results showed that absorption from the injection site, distribution volume, clearance of the PEG-IFN conjugate was significantly slowed down compared to unmodified IFN, which ensured a long circulation of the claimed PEG-IFN conjugate in the blood (more than 14 days).

Example 16. Comparative characteristics of the inventive conjugate PEG-IFN-a2b in comparison with the conjugate PEG-IFN-a2b described in the prototype method.

The structure, the main physicochemical and pharmacokinetic parameters of the inventive PEG-IFN-a2b conjugate are compared with the PEG-IFN-a2b conjugate described in the prototype method (Table 5). The data presented in table. 5, indicate a significant improvement in the properties of the inventive PEG-IFN conjugate in comparison with the conjugate from the prototype method.

Table 5. The main physico-chemical and pharmacokinetic parameters of the inventive conjugate PEG-IFN-a2 in comparison with the conjugate PEG-IFN-a2, described in the prototype method (I8 5951974).

Options PEG-IFN conjugate The inventive conjugate PEG-IFN The conjugate described in the prototype method (and 5 5,951,974) IFN IFN-a2b IFN-a2b PEG structure Linear Linear Molecular Weight of PEG (kDa) twenty 12 The structural formula of the conjugate PEGIFN * Ег Ь bn a1 (a 2b A. n ₃ cc GO thtvGegm a11a-2b Specific activity ^ from native rhIFN 40 28 The number of positional isomers one thirteen Type of bond PEG with protein Stable Unstable (for the main isomer bound to PEG via HH34) Stability in aqueous solutions Stable Unstable Pharmacokinetic parameters The ratio of the area under the curve PEG-IFN / IFN fifty 3-10

* - The structural formula of the PEG-IFN conjugate obtained by the prototype method is given by the link \ UNO Aegid ShogtaEop, 2001, ν. 15, N. 3-4, p. 206.

Example 17. Examples of pharmaceutical compositions based on the inventive conjugate PEG-IFN-a2b.

(BUT) PEG-IFN-a2b according to claim 3 10-500 mcg Sodium Acetate Trihydrate 1 to 5 mg Acetic acid up to pH 5.0 ice Sodium Chloride 8 -9 mg Polysorbate 80 0.01-0.1 mg EDTA disodium salt 0.01 - 0.1 mg Water for injection 1.0 ml (B) PEG-IFN-a2b according to claim 4 10-500 mcg Sodium Acetate 0.4 - 2.0 mg Acetic acid to pH 5.5 Mannitol 50 mg Dextran 15-30 mg Polysorbate 80 0.01-0.1 mg EDT disodium salt A 0.01-0.1 Water for injection 1.0 ml

- 10,020,257

(IN) PEG-IFN-os2b according to claim 4 10-500 mcg Sodium Acetate 0.4 - 2.0 mg Acetic acid to pH 5.5 Mannitol 50 mg Human albumin 10-12.5 mg Polysorbate 80 0.01-0.1 mg EDTA disodium salt 0.01 - 0.1 Water for injections 1.0 ml

Example 18. Packaging in a container of a medicinal product containing PEG-IFN-a2b.

The drug, including an effective amount of the claimed PEG-IFN-a2b (example 17), is poured under sterile conditions at 0.5, 0.8, 1.0 and 1.2 ml (for a dosage of 100 μg / ml), at 0, 5, 0.6, 0.75 and 0.9 ml (for a dosage of 200 μg / ml), 0.5, 0.6, 0.8, 1.0, and 1.2 ml (for a dosage of 300 μg / ml) into syringes of neutral glass of hydrolytic class I, with soldered needles, covered with protective caps, elastic or rigid, sealed with tips on pistons made of butyl rubber laminated with fluoropolymer.

Example 19. Packaging in a container of a medicinal product containing PEG-IFN-a2b.

The drug, including an effective amount of PEG-interferon alpha-2b (example 17), is poured under sterile conditions at 0.5, 0.8, 1.0 and 1.2 ml (for a dosage of 100 μg / ml), at 0, 5, 0.6, 0.75 and 0.9 ml (for a dosage of 200 μg / ml), 0.5, 0.6, 0.8, 1.0, and 1.2 ml (for a dosage of 300 μg / ml) in bottles of neutral glass of hydrolytic class I, corked with fluoro-rubber or butyl-rubber caps with Teflon coating, crimped with aluminum caps.

Example 20. A kit comprising a packaged drug containing PEG-IFN-a2b.

The kit contains 1 or 4 syringes complete with a piston (1 or 4, respectively) / bottle in a blister strip of polymer film, together with instructions for use, which are placed in a pack of cardboard.

Claims (24)

CLAIM 1. A stable conjugate of pegylated interferon-α, which is one positional isomer with monomethoxypolyethylene glycol attached to the Ν-terminal cysteine, of the formula (I) a) g where η is the number of monomer units in a PEG with a molecular weight of from 20 to 40 kDa, with the exception of PEG with a molecular weight of 30 kDa; t is an integer> 4; Ν “Η-ΙΕΝ - interferon-α. 2. The conjugate according to claim 1, in which t is an integer equal to 4. 3. The conjugate according to claim 1, in which interferon-α is a natural or recombinant interferon-a-2b. 4. The conjugate according to claim 1, in which the average molecular weight of polyethylene glycol is 20 or 40 kDa. 5. A pharmaceutical composition having antiviral, antiproliferative and immunomodulating activity, containing a conjugate of the general formula (I) according to any one of claims 1 to 4 in an effective amount and pharmaceutically acceptable auxiliary components. 6. The pharmaceutical composition according to claim 5, intended for use as a medicine for the treatment of viral and oncological diseases and diseases accompanied by primary or secondary immunodeficiency states. 7. The pharmaceutical composition according to claim 6, where the viral disease is hepatitis C or hepatitis B. 8. The pharmaceutical composition according to claim 6, where the cancer is myeloid leukemia. 9. The pharmaceutical composition according to claim 6, where the cancer is a melanoma. 10. A medicament comprising a conjugate of formula (I) according to any one of claims 1 to 4, having antiviral, antiproliferative and immunomodulatory activity. 11. The drug of claim 10, comprising a buffer, an isotonizing agent, a stabilizer, and - 11,020,257 do. 12. The drug according to claim 11, including sodium acetate trihydrate, acetic acid, sodium chloride, polysorbate 80, EDTA, water for injection. 13. The use of the conjugate of formula (I) according to any one of claims 1 to 4 for the manufacture of a medicament having antiviral, antiproliferative and immunomodulatory activity. 14. The use of claim 13 for the manufacture of a medicament having antiviral activity against hepatitis C or hepatitis B. 15. A method of preventing and / or treating viral diseases, comprising administering a therapeutically effective amount of a conjugate of formula (I) according to any one of claims 1 to 4. 16. The method according to clause 15, where the viral disease is hepatitis C or hepatitis B. 17. The method of claim 15, further comprising administering a therapeutically effective amount of ribavirin. 18. A method for the prevention and / or treatment of diseases accompanied by primary or secondary immunodeficiency conditions, comprising administering a therapeutically effective amount of a conjugate of formula (I) according to any one of claims 1 to 4. 19. A method for the prevention and / or treatment of cancer, comprising administering a therapeutically effective amount of a conjugate of formula (I) according to any one of claims 1 to 4. 20. The method according to claim 19, where the cancer is myeloid leukemia. 21. The method according to claim 19, where the cancer is a melanoma. 22. The container is hermetically sealed under sterile conditions, comprising the pharmaceutical composition according to claim 5. 23. The container according to item 22, which is a pre-filled syringe, bottle or auto-injector. 24. A kit comprising a container according to item 23 and instructions for use.