UA99766C2 - NOVEL FUNCTIONALLY ACTIVE, HIGHLY PURIFIED, STABLE PEG-INTERFERON ALPHA CONJUGATE REPRESENTED BY ONE POSITIONAL ISOMER PEG- NaH WITH REDUCED IMMUNOGENITY, PROLONGED BIOLOGICAL ACTION, USEFUL FOR MEDICINAL USE, WITH IMPROVED PHARMACOKINETIC PARAMETERS AND IMMUNOBIOLOGICAL AGENT BASED THEREON - Google Patents

Abstract

The invention relates to novel functionally active, highly purified, stable PEG-interferon alpha conjugate represented by one positional isomer PEG-NH with reduced immunogenity, prolonged biological action, useful for medicinal use, with improved pharmacokinetic parameters, of general formula:, (I)wherein:n – integer values from 227' to 10000, so that molecular weight of PEG amounts to about 10000-40000 Da;m – an integer ≥ 4;IFN – natural or recombinant polypeptide exhibiting IFN-alpha activity,and to immunobiological agent based thereon. The invention further relates to medicinal agents comprising the conjugate of formula (I), pharmaceutical compositions useful for the treatment of viral and oncological diseases, as well as diseases accompanied with primary or secondary immunodeficiency disorders and comprising the PEG-IFN conjugate and therapeutically acceptable adjuvants.

Description

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

Interferons (IFNs) are a group of biologically active proteins or glycoproteins produced by various cells in response to a viral infection or when cells are exposed to certain chemical and biological substances (Izaas5 8 I ipdetap, 1957; Revzika ei ai., 2007). 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 effects of IFN (RezikKa ei ai., 2004;

Vekiv2, 2004).

The genes of various human IFNs were cloned and expressed in bacterial and animal cells, as a result, many recombinant IFNs were obtained and purified (RebikKa ei a!., 2004; Revzika, 2007).

Medicinal preparations containing recombinant IFNs are used in clinical practice for the treatment of a number of viral, oncological and immune diseases (RebzikKa ei ai!., 2004; Spemaiie? 5. RauloivKu, 2009).

Currently, preparations based on IFN are used in clinical practice for the treatment of a number of viral, oncological and immune diseases (Revzika, 2004). IFN drugs are most widely used for the treatment of viral hepatitis (Spemaiie? 5. Rauloi5Ku, 2009), which represent one of the most serious social problems. Currently, there are about 350 million chronic hepatitis B patients and 170 million hepatitis C patients in the world (MagseyPp, 2009). Hepatitis C disease in most cases takes a chronic course with the formation of severe consequences - cirrhosis of the liver and hepatocellular carcinoma (Mod 5. Giapou, 2008). According to WHO data, since 1961 in the USA and Western European countries, chronic hepatitis and liver cirrhosis have moved from 10th to 5th place as a cause of death (MagseyPp, 2009). The use of preparations containing IFN-c for the treatment of a number of leukemias and solid tumors, including recurrent melanoma, is also known (Vykomu»zkKi ei ai., 2002; Oesapigiv ei ai., 2002; Oipa5-Sagdata ei a!., 2006) .

The effectiveness of native IFN preparations is limited by rapid absorption from subcutaneous tissues, large volume of distribution, relatively low stability, short half-life, high immunogenicity and toxicity (UMiY5, 1990). As a result, within a few hours after the injection, a rapid drop in the concentration of IFN in the blood plasma is observed, while interferon is not detected in the plasma already 24 hours after the injection (Spageitsi et al., 1999). A decrease in the concentration of interferon creates conditions for the renewal of virus replication and an increase in the concentration of viral particles (Hat ei ai., 1997). Therefore, there is a need for frequent administration of the drug to achieve effective therapeutic concentrations in the blood plasma, which leads to the occurrence of dose-dependent side effects. As a result of the mentioned features, monotherapy of chronic hepatitis C (CHC) with IFN-a265 for 12 months. led to a stable virological response in only 15-20 Fo patients, in patients with the yiv genotype and a high virological load, the effectiveness of the drug was not observed at all (MeNiispipsop ei ai., 1998).

The therapeutic effectiveness of IFN can be increased when using long-acting drugs, in particular, "PEGylated" IFN (Maptrz ei a!., 2001; Nadluappizv ei ai., 2004; 7ets?et ei ai., 2001).

The production of PEGylated IFN consists in the chemical binding of an interferon molecule with a polymer - monomethoxy polyethylene glycol (MPEG), which consists of repeating subunits of ethylene oxide with a methoxy group at one end and a hydroxyl group at the other. The mPEG molecule can have a different molecular weight and stereochemical structure (linear or branched). To carry out the reaction

PEGylation hydroxyl group at the end of mpPEeEG is activated by various reactive functional groups. Activated mPEG can covalently bind to the protein in one or more places, which depends on the nature of the activated group and the reaction conditions (7aiir5Ku 8 Niggi5, 1997; Kobegiv ei ai., 2002).

PEGylation of IFN leads to an improvement in pharmacokinetics, an increase in the half-life from the blood, a decrease in blood concentration fluctuations, a decrease in immunogenicity and toxicity, an increase in the activity of ip mimo (with a decrease in the activity of ip miigo); increasing stability (siche ei ai., 2000; Kedau ei a!., 2001).

The decrease in activity in the ip myo system directly depends on the molecular weight of the substance used

PEG. The addition of small molecules of PEG with a molecular weight of 5000 Da causes slight changes in the activity of ip migo (Ogase et al., 2005). The pharmacodynamic and pharmacokinetic properties of PEGylated proteins containing PEG with this mass and unmodified proteins practically do not differ.

The addition of PEG with a higher molecular weight and multiple sites leads to a significant decrease in the bioactivity of ip mio, as a result of ineffective binding to the receptor, however, in this case, an improvement in pharmacokinetic and pharmacodynamic properties is observed

of PEGylated proteins (Oyeeiidado ei aili., 1992), which as a result leads to an increase in the biological activity of ip mimo (Vyop 8 Veypoiid, 1998). The form of PEG also affects the bioactivity and pharmacokinetic properties - the conjugate with linear PEG spreads over a larger volume than with PEG, which has a branched structure (Saisei et al., 2003).

Antiviral activity and biological properties of PEG-IFN conjugates also depend to a large extent 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 (A.I. Koregib, 2002) . In the preparation of PEGylated proteins, activated proteins are most widely used

MPEGs capable of binding to free amino groups of proteins (succinimidyl carbonate, succinimidyl succinate, tresylate, triazine, hydroxysuccinimide esters, acetaldehydes, etc.).

Various PEG-IFN conjugates are known from the prior art.

A known conjugate of PEG-IFN-aga with linear PEG with a molecular weight of 1-5000 Da (US patent Mo 5382657, 1995). For conjugation, ethanediol derivatives of MPEG were used, the reaction was carried out at pH-t0 at room temperature for 0.5-4 hours. A 3-fold excess of PEG was used to carry out the IFN PEGylation reaction. Under the conditions used, binding of PEG to IFN occurred through sterically accessible free amino groups of various lysines (IGu5-31, I uz133, I uz134, I uz23,

Gubz131, Guz121, Guvz70, I uz83, I uz49 and Gu 112) with the formation of a urea bond (MopkKaighepP ei ai., 1997). Thus, the obtained PEG-IFN-o2a 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 varied from 6 95 (for I u5112) to 40 95 (for Guz133). The total specific activity of the obtained conjugate, consisting of 11 isomers, was 34 95 of unmodified IFN. The activity of the obtained IP migo conjugates also depended on the molecular weight of PEG and varied from 45 to 95 (for

PEG - 2500 Da) to 25 95 (in the case of PEG - 10000 Da). The disadvantages of the obtained conjugate are: 1. the use of ethanediol-activated mPEG with a low molecular weight (x: 5000 Da), as a result of which the pharmacokinetic parameters of the obtained PEG-IFN conjugate and unmodified IFN differed slightly, in connection with which this the conjugate has not been used in the clinic; 2. formation of a large number of positional isomers with different specific activities, in which PEG was bound to the IFN molecule through free amino groups of various lysines.

Known conjugates PEG-IFN-o2B, obtained by conjugation of native IFN-a260 with linear (patent of Russia

Mo 2311930, 2004) or a branched (Russian patent Meo 2382048, 2008) mPEG derivative with a molecular weight of 13,000-17,000 Da. The reaction was carried out at pH 9.5 for 60 hours (for linear mPEG) or at pH 7.5 for 12 hours (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 95 of the activity of unmodified IFN. Data on bond stability, the number of positional isomers and their specific antiviral activity are not given. The disadvantages of the obtained conjugates are: 1. the use of tresyl derivatives of activated mPEG with a half-life in aqueous solutions of less than 20 min. Earlier, it was shown that during the interaction of tresylate-derivatives of PEG with proteins, in addition to stable amide bonds through amino groups, unstable sulfamate bonds are also formed (sai5 and Virreii, 1995); 2. binding of tresyl-activated PEGs to proteins occurs through all sterically available free amino groups of lysine (Kobegiz et al., 2002), which leads to the formation of several positional isomers; 3. carrying out the IFN PEGylation reaction at high pH values (pH-10) for a long time (60 hours) can lead to a change in the IFN structure; 4. a significant excess of activated PEG during the IFN PEGylation reaction (the PEG/protein molar ratio was 50-100/1) leads to a high cost of the obtained product.

The known conjugate PEG-IFN-a2B6, obtained by binding native IFN-a26 to a branched triazine derivative mPEG with a molecular weight of 7500-35000 Da (Russian patent Mo 2298560, 2004), the total antiviral activity of which was 6.4 95 from activity of unmodified IFN. Data on bond stability, the number of positional isomers and their specific antiviral activity are not provided.

The disadvantages of the obtained conjugate are: 1. the use of triazine-chloride derivatives of activated mPEG, which, in addition to free amino groups, are able to bind to the functional groups of other amino acids - serine, tyrosine, threonine and histidine, which leads to the appearance of a number of isomers, some of which are characterized unstable connection. Moreover, triazine derivatives are currently not used due to their high toxicity (Megopeze 5

Razii, 2005); 2. low specific activity of the PEG-IFN conjugate.

The known conjugate PEG-IFN-c2a, obtained by conjugation of IFN-a2a with branched PEG with a molecular weight of 40 kDa (Russian patent Mo 2180595, 1997). For conjugation, M-hydroxysuccinimide ester derivative MPEG was used, which selectively interacts with available free amino groups of the protein with the formation of a stable amide bond. The reaction was carried out at pH 9 at 4 "C for 2 hours with a ratio of mPEG/protein equal to three. The reaction was stopped and the purification of the obtained conjugate was carried out on the Rgasiode EMO CM 650 (M) sorbent. The yield of the purified conjugate was 40-4595. in this case, an IFN-PEG conjugate consisting of 6 positional isomers was obtained, each of which was connected to one PEG molecule by a stable bond through free amino groups of lysines - Gu531, Gub121, Guz131, Guz134, I uz70 and I uz83 (Vyop et al., 2001; Eozeg et al., 2003). Isomers,

linked to PEG through lysines in position 31, 121, 131 and 134, in total amounted to 94 95. The activity of the total conjugate, consisting of 6 positional isomers, was 1-7 95 from native IFN-sg2a (Vaiyop ei ai ., 2001; Vosheziip ei ai., 2006). Despite the low antiviral activity of ip migo, this conjugate compared to unmodified IFN had improved pharmacokinetic properties (5iima ei ai., 2006,

Voshevziip ei ai., 2006), and is currently used in the clinic under the name "Pegasis" (manufactured by the company "Hoffmann-La Roche").

The disadvantages of the obtained conjugate are: 1. the use of M-hydroxysuccinimide-ester of activated PEG, which binds to all sterically accessible amino groups, as a result, the obtained conjugate represented a mixture of 6 isomers differing in specific activity. 2. low antiviral specific activity of the received ip myo conjugate.

The prototype of this invention is the PEG-IFN-sa2r conjugate, described in US patent No. 5951974, 1999).

For the PEGylation reaction, linear mPEG activated by a succinimidyl carbonate group (5C-TRES) with a molecular weight of 5000 to 12000 Da 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" (the company "ZPPegipd-Riosdp", USA).

It was established that the obtained PEG-IFN-c26 conjugate (the "Pegintron" drug) consists of 13 positional isomers, each of which contains one PEG molecule attached to different parts of the protein molecule (U/apod ei ai. 2000; Sgase ei ai ., 2001; Moipdvieg eyi ai.,, 2002). It was shown that the PEG molecule was bound to interferon not only through free amino groups of lysines (I u531 Guz 49, I uz83, I ug121,

Guz131, Guz133, Gub134 and I uz164) and M-terminal cysteine (Subi), but also through the imidazole ring of histidine (Niz7, Niz34), tyrosine (Tui129) and the OH group of serine (5eg163). The content of individual isomers varied from 0.8 (Tupt29) to 47.95 (Nikh34). The antiviral activity of the total preparation of Pegintron, consisting of 13 positional isomers, was 2895 times that of the native protein. The specific activity of individual isomers varied from 11 to 37 95 of the native IFN-a2b (Uoipozgeg ei ai., 2002). The highest specific activity was in the main isomer conjugated with PEG through Niz34, which was 37 95 of the native protein.

The free amino groups of IFN were linked to PEG through a stable bond, whereas in the major isomer, approximately 4795, PEG was linked to the imidazole ring of histidine (Niz34) through an unstable (in aqueous solutions) carbamate bond ( Korbegiz ei ai!., 2002).

Pharmacokinetic parameters of the obtained conjugate were better than those of unmodified IFN (5iima eia!., 2006).

The disadvantages of the resulting conjugate are: 1. the use of succinimidyl carbonate activated PEG, which binds not only to free amino groups of IFN, but also to functional groups of other amino acids. As a result, the resulting conjugate consisted of a mixture of 13 positional isomers differing in specific antiviral activity; 2. the formation of an unstable imidazole-carbonate (carbamate) bond in the main (Nivb-34) isomer of the PEG-IFN conjugate leads to the fact that when the drug "Pegintron" enters the body, it is hydrolyzed and the native IFN is released, which has a biological effect . Thus, the drug "Pegintron" is best regarded as a pro-drug, which, after being injected into the body, is cleaved with the formation of active interferon (Rozieg, 2004). 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 task of this invention was to obtain a new stable conjugate of PEGylated IFN, with IFN alpha activity, consisting of one positional isomer, with improved stability, with reduced immunogenicity, with improved pharmacokinetic parameters, with an optimal combination of PEG molecular weight parameters and antiviral activity. suitable for the creation of a medicinal drug for medical purposes, which contributes to the expansion of the arsenal of drugs of a given focus, a pharmaceutical composition based on the obtained PEG-IFN.

The task is solved by creating a new functionally active conjugate molecule

PEG-IFN with interferon-alpha activity, in which a linear PEG molecule with a molecular weight of 10 to 40,000 Da is linked to an IFN-sa molecule by a stable bond strictly through the alpha amino group of the M-terminal cysteine so that the general formula of the conjugate PEG-IFN with linear mPEG is the following compound (1):

SN, I o-sny-snA-ro I snd-k "NA -IEM (I), p t where: p - whole values from 227 to 10000; t - integer 24;

IEM is a natural or recombinant polypeptide that has IFN-alpha activity.

In the resulting conjugate, linear PEG with a molecular weight of 10,000-40,000Da is connected to the alpha amino group of the M-terminal amino acid of IFN.

The selectivity of IFN modification strictly according to the alpha-amino group of the M-terminal amino acid is achieved by using aldehyde-activated mPEGs of the general formula (II) and carrying out the reaction

PEGylation at рНеб: sn. I o-sny-sn-o Clearly (I) p t1 nn where: p - whole values from 227 to 10,000, so that the molecular weight of PEG is approximately 10,000-40,000 Da; t is an integer of 24.

The optimal ratio of PEG molecular weight parameters and antiviral activity is achieved due to the use of activated mPEG (II) with a value of tg24.

Reduction of immunogenicity, toxicity and improvement of pharmacokinetic parameters is achieved by increasing the molecular weight of the attached PEG.

The following are new compared to the prototype: 1. aldehyde derivatives of activated mPEG were used to obtain the PEG-IFN conjugate; 2. structural formula of PEG-IFN conjugate; 3. the obtained PEG-IFN conjugate is represented by one positional isomer; 4. stable connection 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.

Recombinant human IFN-c20 (manufactured by CJSC "Biokad") and butyric or pentaalaldehyde derivatives of mPEG of formula (II) with a molecular weight of 20,000 Da were used to obtain the claimed PEG-IFN conjugate.

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 molar ratio of PEG/protein was 2.5-5/1. Control of the formation of the PEG-IFN conjugate was performed using polyacrylamide gel electrophoresis (PAAG) in the presence of sodium dodecyl sulfate (SDS) under reducing conditions and reversed-phase high-performance chromatography (3F HPLC). Purification of monoPEG-IFN from reaction products, unmodified IFN, and unwanted forms of PEG- IFN containing two or more PEG molecules per protein molecule was performed using chromatography on cation-exchange sorbents. The elution of mOonNOoPEG-IFN was carried out with a gradient of sodium chloride concentration from 0.05 to 0.2 g in a buffer solution with a pH below 6. Purified monoPEG preparation -IFN was dialyzed against 10-50 mM buffer solution with pH 4-5, salts or polysaccharides, or polyhydric alcohols, or polyvinylpyrrolidones, or monosaccharides, or aminosugars, or proteins, or amino acids and nonionic dete were added rgents and stored in plastic or glass vials with a siliconized surface at a temperature of 4:52 "C.

To characterize the obtained monoPEG-IFN-o2or conjugate, its purity, homogeneity, physicochemical, biological, and pharmacokinetic parameters were investigated in comparison with the unmodified

IFN

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

Fig. 1. Kinetics of the formation of the IFN-o2p conjugate during the reaction using the butyraldehyde derivative of MPEG.

Fig. 2. Reverse-phase HPLC of the PEG-IFN-o2B5 conjugate on the Zutteigu S18 column (4.6 x 150 mm).

Fig. 3. Electrophoretic analysis of the PEG-IFN-o2B conjugate in comparison with unmodified IFN-c26.

Fig. 4. MAGO mass spectra of unmodified IFN-c265 and PEG-IFN-o2b conjugate.

Fig. 5. Localization of the PEGylation site in the PEG-IFN-c2B conjugate. Comparison of mass spectra of tryptic peptides of unmodified IFN-co2b (A) and PEG-rcIFN-a2 conjugate (U) in the range of t/2 1200-1400.

Fig. 6. Thermostability of PEG-IFN-O2 conjugate.

Fig. 7. Proteolytic stability of the PEG-IFN-a25 conjugate upon treatment with trypsin.

Fig. 8. Immunogenicity of the PEG-IFN-a2B conjugate compared to unmodified IFN-a2b.

Fig. 9. Pharmacokinetics of the PEG-IFN-a25 conjugate in comparison with unmodified IFN-a26.

Examples of a specific implementation of the method of obtaining PEG-IFN-o2oB and studying its properties are given below.

PEG-IFN-a2 conjugates of formula (I), which are the object of this 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 value of antiviral activity. Also, pharmaceutical compositions containing as an active ingredient an effective amount of the conjugate of formula (I) and obtained by known methods used in pharmaceuticals can be used both separately and together with other therapeutic agents (for example, ribavirin) for prevention and/ or treatment of viral diseases (chronic active hepatitis (in particular,

hepatitis B and C) (Dau et al., 2006; MeNiispizop et al., 2009). The object of this invention is also a method of prevention and/or treatment of viral diseases, for example, hepatitis C and hepatitis B, which includes the introduction of a therapeutically effective amount of the declared conjugate of formula (1).

The use of IFN-γ and PEGylated interferons-α as immunomodulators for the treatment of oncological diseases, in particular, hairy cell leukemia, laryngeal papillomatosis, melanoma, renal carcinoma, myeloid leukemia, Kaposi's sarcoma, is also known from the state of the art (Oesaigi5 ei ai., 2002; VykomuvkKi ei ai !., 2002; Oipiaz-Saghdata, 2006; I odtsai, 2008; KayenNieg, 2010). On the basis of the etiopathogenesis of these diseases known from the state of the art and the use of IFN-α, as well as conjugates for their prevention and/or treatment, the objects of this invention are also medicinal products and pharmaceutical compositions containing the claimed conjugate PEG- IFN-Yai in an effective amount, having an antiproliferative and immunomodulatory effect, which can be used for the prevention and/or treatment of oncological diseases and diseases accompanied by primary or secondary immunodeficiency states, as well as the object of this invention is a method of prevention and/or treatment oncological diseases and diseases accompanied by primary or secondary immunodeficiency states, which includes the introduction of a therapeutically effective amount of the conjugate PEG-

IFN-a formula (1).

The conjugate of formula (I) with an optional pharmacologically acceptable filler, diluent and Mabo pharmaceutically acceptable excipients is administered intravenously, subcutaneously, intramuscularly or in any other suitable way, for example, in the form of capsules, syrup, spray, drops, etc. injection or suppository. The route of administration is changed depending, for example, on symptoms and age, the frequency of administration and the interval between administrations are changed depending on the disease and its severity or on the purpose (therapeutic or prophylactic use), the effective amount of the active beginning of PEG-IFN-th is selected taking into account the above factors

As pharmaceutically acceptable auxiliary components that can be included in a pharmaceutical composition with PEG-IFN-a2, you can use: buffer salts (for example, acetate, citrate, hydrogen carbonate, phosphate buffers), stabilizers (for example, polysorbates, EDTA, polyvinylpyrrolidone, Dextrans, CSA, paraoxybenzoic acid esters, alcohols (e.g. benzyl alcohol), phenols, sorbic acid), disinfectant component (e.g. benzyl alcohol), osmotic pressure regulator (e.g. sodium chloride, potassium chloride, polyols, e.g. glycerin , arabitol, sorbitol, »mannitol, lactose, dextran), surfactants (for example, ChSA, polyvinylpyrrolidone, lecithin, Tween 80, polyoxyethylene-polyoxypropylene copolymer, casein), surfactants (for example, block copolymers of ethylene oxide and propylene oxide, propylene oxide and ethylene oxide, sorbitan monolaurate, sorbitol ester, polyglycerin fatty acid ester, cocamide OEA lauryl sulfate, alkanolamide, polyoxyethylene propyl stearate lenglycol, polyoxyethylene lauric ether, polyoxyethylene cetyl ether, polysorbate, glycerol monostearate, glycerol distearate, sorbitol monopalmitate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate and propylene glycol monostearate), antioxidants (for example,

Trilon B, I-cysteine, sodium sulfite, sodium ascorbate, glutathione, 2-mercaptoethanol, dithiotriitol, cysteine hydrochloride monohydrate, ascorbic acid) and diluents (eg, water).

Example 1

Preparation of the PEG-IFN-a2b conjugate using the butyraldehyde derivative of MPEG

To 785 ml of a buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.0) containing recombinant IFN-α2 at a concentration of 1.7 mg/ml, 16 ml of a 1 M solution of sodium cyanoborohydride was added, mixed and 5 g of dry butyraldehyde PEG derivative with a molecular weight of 20,000 daltons. The mixture was thoroughly mixed and incubated for 22 hours at a temperature of 1753 "C. After different intervals of time, aliquots of 50 μl were taken from the reaction mixture and the kinetics of the formation of the PEG-IFN-α2 conjugate was analyzed using the electrophoresis method in PAGE in the presence

VAT To do this, 1/3 of the buffer volume containing 125 mM Tris-HCl, pH 6.8, 95 glycerol, C 96 DDS, 0.005 95 bromophenol 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 at 12.5 95

PAAG in the presence of VAT. After electrophoresis, the gel was stained with Coomassie K-250.

The kinetics of PEG-IFN-o2 conjugate formation is shown in Fig. 1. At the time when the content of PEG-IFN was more than 70 95, the reaction mixture was diluted 10 times with 5 mM sodium acetate buffer, pH 5.0.

Example 2

Preparation of the PEG-IFN-a265 conjugate using the pentaaldehyde derivative of MPEG

To 100 ml of a buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5) containing recombinant IFN-4250 at a concentration of 2.2 mg/ml, 2.05 ml of a 1 M solution of sodium cyanoborohydride was added, mixed and added 0.9 g of dry pentaldehyde PEG derivative with a molecular weight of 20,000 daltons. The mixture was thoroughly mixed and incubated for 24 hours at a temperature of 622 °C. After different intervals of time, aliquots of 50 μl were taken from the reaction mixture and the kinetics of the formation of the PEG-IFN conjugate was analyzed using the PAGE electrophoresis method, as described in example 1. at the time when the content of PEG-IFN was more than 70 95, the reaction mixture was diluted 10 times with 5 mM sodium acetate buffer, pH 5.0.

Example C

Purification of conjugate monoPEG-IFN-YA25 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) equilibrated with 5 mM sodium acetate buffer, pH 5.0 (buffer A) at a rate of 5 ml/min. The column with the sorbent was washed sequentially with buffer A and buffer A containing a Mass concentration gradient from 0.05 to 0.2 M. Aliquots were taken from each fraction for sample analysis by electrophoresis in PAAG, as described in example 1. Fractions containing monoPEGylated PEG-IFN-a2 conjugate was combined, dialyzed against 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, sterile filtered and stored at a temperature of 42 26.

Example 4

Purification of conjugate monoPEG-IFN-YA25 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 (5P-Sepharose, 50 ml) equilibrated with 5 mM sodium acetate buffer, pH 5.0 (buffer A) at a rate of ml/min. The column with the sorbent was washed sequentially with buffer "A" and buffer A containing a Mass concentration gradient from 0.03 to 0.3 M. From each fraction, aliquots were taken for sample analysis by electrophoresis in PAGE, as described in example 1. Fractions that contain a monoPEGylated conjugate

PEG-IFN-α2 was combined, dialyzed against 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, sterile filtered and stored at a temperature of 4°C.

Example 5

Analysis of the PEG-IFN-α26 conjugate using the reverse-phase high-performance liquid chromatography method

The PEG-IFN-o2 conjugate, obtained according to item 3, was diluted with 20 mM sodium acetate buffer, pH 5.0, to a concentration of 0.1 mg/ml, and 100 μl of the obtained sample was applied to a Zutteig C18 column (4.6 x 150 mm).

The research was carried out on a "Vgeele" chromatograph of the MUMaieg company at 214 nm. From the results presented in fig. 2, it follows that, according to ZF HPLC, the purity of the declared PEG-IFN-a2 conjugate is more than 99 95.

Example 6

Determination of the level of endotoxins

The content of bacterial endotoxins (BE) in samples of the PEG-IFN-a2 conjugate prepared by Zip. 4, IMP was determined using the LAL test (modified gel-thrombus test) in accordance with the requirements of ZFS 42-0002-00. In the work, a diagnostic multicomponent set of Avzosia (e5 oi SARE) was used

SOY, Ips. LAL reagent RUKOTEI IF with a sensitivity of 0.03 EE/ml, endotoxin control standard (CSE, 0.5 μg in a vial), water for the LAL test. From the results presented in the table. 1, it can be seen that the content

BE in conjugate samples is less than 1.5 units of endotoxin (OE) per mg of protein, which is much lower than the level of BE acceptable for medical preparations based on recombinant proteins.

Table 1

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

Example 7

Electrophoretic analysis of the PEG-IFN-sa2B conjugate in comparison with unmodified IFN-saB

The PEG-IFN conjugate sample obtained according to item 3 was analyzed by electrophoresis in PAGE as described in item 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 Kumasi K-250 dye. At the same time, electrophoresis of unmodified IFN-α2 was performed. The PEG-IFN conjugate was represented by one band (Fig. C, track 1). From the electrophoregrams of the PEG-IFN-o2 conjugate and unmodified IFN-a2, presented in figure 3, it is clear that the PEG-IFN-a conjugate has a higher molecular weight than rchIFN-a (Fig. C, tracks 1 and 2).

It should be noted that the exact value of the molecular weight for PEGylated proteins cannot be determined by the EF method, because the attachment of the hydrophilic PEG molecule to the protein significantly increases the radius

Stokes of the resulting complex. As a result, the advancement of the PEG-protein complex in the gel slows down, and the value of its molecular weight turns out to be much higher than the sum of the molecular weights of the protein and PEG.

Example 8

Determination of the molecular weight of the PEG-IFN-o2 conjugate in comparison with unmodified IFN-c2 by the mass spectrometer method of 1 μl of the PEG-IFN conjugate sample obtained according to item 3 and 0.3 μl of 2,5-dihydroxybenzoic acid solution (Ayagis, 10 mg x ml" in 20 95 acetonitrile in water with 0.595 TFO) were mixed and dried in air. A sample of unmodified IFN-a2 was prepared in a similar way.

Mass spectra were obtained on the MAG OiI time-of-flight mass spectrometer OKgayeh I! VKOKEK (Germany), equipped with a UV laser (Ma). Mass spectra were obtained in the mode of positive ions, in the linear mode, the error of the measurement of the average mass does not exceed 10-15 Da.

The results of the mass spectrometric analysis of unmodified rchIFN-o2 and the PEG-IFN-o2 conjugate in the range of t/7 from 10,000-50,000 are shown in Fig. 4. From fig. 4A shows that the mass spectrum of rchIFN-o2 contains the main peak corresponding to a singly charged ion (MI" with a molecular weight of 19295 Da.

In the mass spectrum of the PEG-IFN-o2 conjugate shown in fig. From B, a blurred main peak corresponding to the singly charged MI" ion with a molecular mass of 40498 Da is presented. The blurring of the peak is associated with the heterogeneity of monomethoxyPEG preparations. For the PEG-IFN conjugate, the measured value of t/: in the maximum of the peak (40498 Da) coincides with calculated data on the sum of the molecular masses of IFN-o2 (19,295 Da) and attached PEG (20,000 Da).

Example 9 Localization of the PEGylation site in the H-IFN-o2b conjugate

To 5 μl of the PEG-IFN conjugate sample obtained according to item 3, 5 μl of a solution of modified trypsin (Rgoteda) in 0.05 M MNANSO:" with a concentration of 15 μg x ml was added." Hydrolysis was carried out for 16 hours at 37 " C, then 10 μl of 0.5 95 TFO in a 10 95 solution of acetonitrile in water was added to the solution and mixed thoroughly. A sample of unmodified IFN was prepared in a similar way. The resulting solutions were used to obtain MAG OI mass spectra.

The localization of the binding site of PEG with the IFN molecule was determined by comparing the mass spectra of the tryptic hydrolyzate of the PEG-IFN-o2 conjugate and unmodified rchiIFN-a2. In the tryptic hydrolyzate of the PEG-IFN-a2 conjugate, the peptide corresponding to the part of the protein on which the modification took place must be absent. In the table 2 presents the mass spectra of experimental tryptic peptides for unmodified IFN-Yai and PEG-IFN-42 conjugate. The table shows that the mass spectra of tryptic peptides of unmodified IFN-425 practically coincide with the mass spectra of tryptic peptides of the PEG-IFN-a2 conjugate (Table 2), but the peptide is missing in the tryptic digest of the PEG-IFN-a2 conjugate with a molecular weight of 1313.649, which is present in a tryptic digest of unmodified rchIFN-α2 (see also Fig. 5). According to the analysis of the theoretical masses of tryptic IFN-a2 hydrolyzate, the peak with t/7 1313.649 corresponds to the M-terminal peptide of the protein (Table 2). Its absence in the tryptic digestion of the PEG-IFN-o2 conjugate indicates a modification of the M-terminal peptide, which, having become heavier per mass of PEG, is outside the studied range of the spectrum. Binding of PEG to the IFN molecule took place via the only free amino group present in this peptide - via the amino group M- of the terminal cysteine.

Table 2

Experimental mass spectra of tryptic peptides of the PEG-IFN-α2 conjugate and unmodified

IFN-a2 in comparison with theoretical values

Experimentally discovered peptides of 131349 | -:B - 1/ 1313.6266 | 1-12 |SOIROTNIOVZAI G-Z:Z

Example 10 Determination of the specific antiviral activity of the PEG-IFN-c2p conjugate

Antiviral specific activity was studied in the samples obtained according to point C and point 4 according to the protocol described in the European Pharmacopoeia using a culture of transfected cells, MVOK, sensitive to alpha-type interferon and vesicular stomatitis virus (M5M). In the table The results of the study of antiviral activity are presented. The international reference standard was used as a standard. From the table It can be seen that, despite the addition of a PEG molecule with a molecular weight of 20,000 Da, the antiviral activity of the obtained conjugates is 42-45,905 times 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 12,000 Da had lower activity (28-30 9).

Table C

The specific antiviral activity of the PEG-IFN-α2 conjugate in comparison with unmodified IFN-α2 to the activity of unmodified my P roi

Example 11 Study of the thermal stability of the PEG-IFN-a conjugate 25 ml of the sample of the PEG-IFN conjugate obtained according to item 3 was placed in a water bath at a temperature of (50:-2)7С and after different time intervals the appearance of turbidity in the solution was investigated protein, measuring the optical density at 340 nm. Thermostability of unmodified IFN-a2 was studied similarly.

The formation of insoluble protein aggregates led to the appearance of turbidity and an increase in optical density at 340 nm. In fig. 6 presents the results of the study. During the observation period (28 hours), the PEG-IFN-α42 conjugate remained stable, while the unmodified IFN-α25 almost all precipitated after 28 hours.

Thus, the obtained data allow us to draw a conclusion about a significant increase in the thermal stability of the PEG-IFN-a2 conjugate compared to the unmodified protein.

Example 12

Study of the proteolytic stability of the PEG-IFN-o260 conjugate when treated with trypsin

To 1 ml of the PEG-IFN conjugate obtained according to item 3, 150 μl of M Tris-HCI buffer, pH 8.5, 2 μl of 0.5 M solution of Sas" and 15 μl of trypsin (Rgoted) with a concentration of 20 μg were added / ml. Samples were incubated at 37 "C. After certain time intervals, aliquots of 100 μl were taken and 150 μl of 0.5 95 trifluoroacetic acid solution was added to stop the reaction. Samples containing unmodified

IFN-a2. The samples were then analyzed by reverse-phase HPLC and the area of the main peak was determined. The results of the study shown in Fig. 7, show that upon treatment with trypsin, the proteolytic stability of the PEG-IFN-α2 conjugate is higher than that of unmodified IFN-α26.

Example 13 Determination of the stability of the PEG-IFN-a265 conjugate during storage

From the sample obtained according to point 3, aliquots were taken into sterile tubes with lids, of the "Eppendorf" type.

The protein concentration in the PEG-IFN-42 conjugate was 1 mg/ml. The samples were placed in a refrigerator at a temperature of (b-2)"C. After certain time intervals, samples were taken and the specific antiviral activity and homogeneity of the drug were analyzed using ZF HPLC and electrophoresis (EF) in PAGE in the presence of DDS under non-reducing conditions, as described in point 1.3 of the data in Table 4, it can be seen that when stored at a temperature of (6:22)"C, the PEG-IFN-a2 conjugate is stable for at least 24 months.

Table 4

The stability of the PEG-IFN-o26 conjugate at temperature (622370 goals Go 13 1 6 1 9 1 12 1 18 | 24

Homogeneity, 90

You are too bad |for sao soe | ol zol

Homogeneity, 9o

Example 14 Study of the immunogenicity of the PEG-IFN-a2B conjugate

The PEG-IFN conjugates obtained according to item C and item 4 were diluted to a concentration of 5 million IU/ml and injected 200 μl (1 million IU/mouse) intramuscularly into ISK mice with a body weight of 20 22 g once a week for 5 weeks (5 groups of 5 mice per group). In parallel, a sample of unmodified IFnN was prepared and administered to mice in a similar manner, also in a dose of 1 million. At the end of the fifth week, blood was taken from the animals using a retroorbital puncture. Blood serum was obtained by a standard method. Titer of antibodies in sera obtained as a result of immunization of mice with unmodified IFN and conjugates

PEG-IFN was determined using a direct solid-phase enzyme-linked immunosorbent assay (ELISA). For this, 100 μl of a solution of unmodified IFN-42 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 two-fold serial dilutions. Anti-mouse immunoglobulins conjugated with peroxidase were used to detect the formed immune complex. Antiserum titer 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-α2 conjugates is 8 times lower than that of unmodified IFN.

Example 15 Study of the pharmacokinetics of the conjugate PEG-IFN-a2r

A sample of the PEG-IFN conjugate obtained according to example 3 in a dose of 1 million IU was administered intraperitoneally to male mice. In parallel, another group of mice was injected with unmodified IFN-α2.

After certain time intervals, blood was collected from the animals by means of retroorbital puncture. Blood serum was obtained by a standard method. The presence of interferon in serum was determined by antiviral activity. The results of pharmacokinetics are presented in fig. 9. The results show that the declared PEG-IFN-42 conjugate has a significant prolonged effect. When native IFN-a2 was administered, its content in the blood of animals reached a maximum 1 hour after administration, and then decreased very quickly. When the PEG-IFN conjugate was administered, the maximum content of IFN in the blood of animals was observed after 12 hours (Fig. 9), and this level was maintained for 50 hours, and only then there was a slow decrease in the content of IFN-a2 during 350 hours.

The area under the curve (AUC) for the claimed PEG-IFN conjugate was more than 50 times higher than the corresponding value for unmodified IFN. It should be noted that for the PEG-IFN-α2p conjugate described in the prototype method, bound to PEG with a molecular weight of 12,000 Da, the value of AOS exceeded the value of AOS for IFN by only 3-10 times.

Based on the obtained results (Fig. 9), the main pharmacokinetic parameters for the claimed PEG-IFN-o2 conjugate were calculated. The results showed that the absorption from the injection site, the volume of distribution, and the clearance of the PEG-IFN conjugate were significantly slowed down compared to unmodified IFN, which ensured long-term circulation of the declared PEG-IFN conjugate in the blood (more than 14 days).

Example 16

Comparative characteristics of the declared conjugate PEG-IFN-c2B in comparison with the conjugate PEG-

IFN-a2B0, which is described in the prototype method

A comparison of the structure, main physicochemical and pharmacokinetic parameters of the claimed PEG-IFN-a25 conjugate was made in comparison with the PEG-IFN-sa2r conjugate described in the prototype method (Table 5). The data presented in the table. 5, indicate a significant improvement in the properties of the claimed PEG-IFN conjugate compared to the conjugate from the prototype method.

Table 5

The main physicochemical and pharmacokinetic parameters of the declared conjugate

PEG-IFN-o2 in comparison with the PEG-IFN-a2 conjugate described in the prototype method (US patent Mo 5951974)

The conjugate described in method-

The declared conjugate PEG-IFN prototype (US patent Mo 5951974)

Molecular weight

The structural formula of the interferon ee ac X conjugate of PEG-IFN"U Z alfagr NS o; Interferon alfa 2r

Specific activity, 90 isomers

The type of connection of PEG with stable Y Unstable (for the main bicom stable isomer connected to PEG through

Niz34 solutions

The ratio of AOS PEG-x- The structural formula of the PEG-IFN conjugate, obtained by the prototype method, is given by reference "MNO Ogyd Ipgoptaiop", 2001, m. 15, M. 3-4, p. 206.

Example 17

Examples of pharmaceutical compositions based on the claimed conjugate PEG-IFN-a2r (A)

PEG-IFN-a2B according to paragraph C 10-500 μg

Sodium acetate trihydrate 1-5 mg

Glacial acetic acid to pH 5.0

Sodium chloride 8-9 mg

Polysorbate 80 0.01-0.1 mg

EDTA disodium salt 0.01-0.1 mg

Water for injections 1.0 ml (B)

PEG-IFN-o2B according to item 4 10-500 μg

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

EDTA disodium salt 0.01-0.1

Water for injections 1.0 ml (B)

PEG-IFN-o2B according to item 4 10-500 μg

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-a2r

The medicinal product, which includes an effective amount of the declared PEG-IFN-a25 (Example 17), is bottled under sterile conditions in 0.5, 0.8, 1.0 and 1.2 ml (for a dosage of 100 μg/ml), in 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 each (for a dosage of 300 μg /ml) in syringes made of neutral glass hydrolytic class, with soldered needles, covered with protective elastic or hard caps, closed with tips on a piston made of butyl rubber laminated with fluoropolymer.

Example 19 Packaging in a container of a medicinal product containing PEG-IFN-c2b

The medicinal product, which includes an effective amount of PEG-interferon alfa-25 (Example 17), is bottled under sterile conditions in 0.5, 0.8, 1.0 and 1.2 ml (for a dosage of 100 μg/ml), in 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 each (for a dosage of 300 μg/ml) in vials made of neutral glass of the I hydrolytic class , closed with fluororubber or butyl rubber stoppers with Teflon coating, crimped with aluminum caps.

Example 20

A kit including a container-packaged medicinal product containing PEG-IFN-a25

The set contains 1 or 4 syringes complete with a piston (1 or 4, respectively)/vial in contour cell packaging made of polymer film together with instructions for use, which are placed in a cardboard pack.

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Time (hours) "it, 8 Pharmacokinetics of the extract of PER-FN-aOb is comparable to unmodified FEV

Claims (25)

1. Stable conjugate of PEGylated interferon-y of the formula (I), which is one positional isomer of SNU исн. 227 to 10000; t - integer 2 4; M'N-ieM. interferon-a. 2. Conjugate according to claim 1, in which t is an integer equal to 4. 3. The conjugate according to item I, in which interferon-i is natural or recombinant interferon-a-2b. 4. The conjugate according to claim 1, in which the average molecular weight of polyethylene glycol is from 10 to 4А4OkDa. 5. Conjugate according to claim 1, in which the M-terminal group is represented by a cysteine residue (Sub). b. A pharmaceutical composition having antiviral, antiproliferative and immunomodulatory activity, containing a conjugate of the general formula (I) in an effective amount and pharmaceutically acceptable auxiliary components. 7. Pharmaceutical composition according to point b for use as a medicinal product for the treatment of viral and oncological diseases and diseases accompanied by primary or secondary immunodeficiency states. 8. Pharmaceutical composition according to claim 7, where the viral disease is hepatitis C or hepatitis B. 9. Pharmaceutical composition according to claim 7, where the oncological disease is myeloid leukemia. 10. Pharmaceutical composition according to claim 7, where the cancer is melanoma. 11. Medicinal product including the conjugate of formula (1) having antiviral, antiproliferative and immunomodulatory activity. 12. The drug according to claim 11, which includes a buffer, an isotonizing agent, a stabilizer, and water. 13. Medicinal product according to claim 12, which includes sodium acetate trihydrate, acetic acid, sodium chloride, polysorbate 80, EDTA, water for injections. 14. Use of the conjugate of formula (I) for the preparation of a medicinal product having antiviral, antiproliferative and immunomodulatory activity. 15. Application according to claim 14 for obtaining a medicinal product having antiviral activity against hepatitis C or hepatitis B. 16. A method of prevention and/or treatment of viral diseases, in which a therapeutically effective amount of the conjugate of formula (1) is administered. 17. The method according to claim 16, where the viral disease is hepatitis C or hepatitis B. 18. The method according to claim 16, in which a therapeutically effective amount of ribavirin is additionally administered. 19. A method of prevention 1/or treatment of diseases accompanied by primary or secondary immunodeficiency states, in which a therapeutically effective amount of the conjugate of formula (1) is administered. 20. A method of prevention and/or treatment of oncological diseases, in which a therapeutically effective amount of the conjugate of formula (1) is administered. 21. The method according to claim 20, where the oncological disease is myelogenous leukemia. 22. The method according to claim 20, where the cancer is melanoma. 23. A container hermetically sealed under sterile conditions suitable for storage before use, which includes the pharmaceutical composition according to claim 6. 24. The container according to claim 23, where said container is a pre-filled syringe, vial or auto-injector. 25. A set including a container according to claim 24 1 instructions for use. Tve ipuepnop heiYage5 yu pohue!i GppsyopaPu asiue, Pigu rygiNei, "gabie RES-ipgepegop aiYirva sop)irage hergezepteid bu ope roziyopai! isoteg RES-MUN myth tedisei ittiporepigu, rgoYopreai BioiIorisai aspop, iveyi! Gog tedisipai ive, well itrgouei rragtasoKipeis ragateget", oh repega! Tgtia: snu i- sn.-sn.-o Ya svd-k hN-- EM p t mkV) huVegeip: p - ipgereg uaishev izhygot 227 tyu 10000, vo vag toIesshag huei and oi RES atoipiv tyu aroshi 10000-40000 Ba; t - ap sh(ereg 2 4; IEM - pashgai og tesotbripapi roYIurerinde ekhvirbtsipe TEM -aIrvba asnuu, apa syu ittiporbioIorisai arepi razey Fegeop. That ipueptsop TgSheg geYage5 then tedisipa! arepi5 sotrgivipe She sop)yragse oi Togtsha (I), rrpagtaseshisa! sotroviyope iveyi og She yteantepi oi higa! apa opso1Iorisai dizeazev, av me av dizeave5 assotrapigya hYyV rgitagu og zesopdagu ittipodeisiepsu dizogdete apa sotrgivip" pe REO-IEM sop)yragse apa "pegareshisaPu asseriarie aduiuapv.