KR20130056885A - The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer - Google Patents

Abstract

The present invention relates to the pharmaceutical industry and pharmaceuticals, in particular novel PEG-interferon derivatives and novel polyethylene glycols and interferons represented by formula (I), which have interferon alpha activity, reduced immunogenicity, prolonged biological effects and improved pharmacokinetic parameters. Relates to the discovery of functionally active, high stability conjugates, wherein n is an integer from 227 to 10000 resulting in a molecular weight of PEG of about 10000 to 40000 Da; m is an integer of ≧ 4; IFN is an IFN-α Active natural or recombinant polypeptide.] The invention also provides for the treatment of drugs, viral infections and cancers, as well as diseases associated with primary or secondary immunodeficiency, containing the claimed conjugate represented by formula (I). A pharmaceutical composition comprising a therapeutically acceptable excipient and a PEG-IFN conjugate suitable for the present invention. Preventing and / or treating a disease associated with primary or secondary immunodeficiency, including the use of formula (I) for use in drugs with immunomodulatory activity, the administration of a therapeutically effective amount of a conjugate represented by formula (I) To a container having said pharmaceutical composition:
(I)

Description

THE NEW STABLE POLYETHYLENE GLYCOL CONJUGATE OF INTERFERON ALPHA, REPRESENTED BY ONE POSITIONAL ISOMER}

The present invention relates to a medicament, ie a physiologically active conjugate of interferon, and more particularly to a novel conjugate of interferon and polyethylene glycol (PEG) which can be used in medicine, for example viral diseases, immunological diseases and oncological diseases. .

Interferon (IFN) is a group of biologically active proteins or glycoproteins produced by various cells in response to viral infections or as a result of the effects of certain chemical and biological substances on cells (Issacs & Lindeman, 1957; Pestka et. al., 2007). Conjugation of cell receptors with IFNs results in the induction of a number of intracellular proteins that mediate antiviral, immunomodulatory and antiproliferative IFN effects (Pestka et al., 2004; Bekisz, 2004).

Genes of several human IFNs have been cloned and expressed in bacterial and animal cells, and as a result, many recombinant IFNs have been developed (Pestka et al., 2004; Pestka, 2007). Drugs containing recombinant IFNs are used in clinical practice to treat many viral, oncological and immunological diseases (Pestka et al., 2004; Chevaliez & Pawlotsky, 2009).

Currently, drugs based on interferon are used in clinical practice to treat many viral, oncological and immunological diseases (Pestka et al., 2004). IFN drugs are most widely used for the treatment of viral hepatitis, one of the most serious social problems (Chevaliez & Pawlotsky, 2009). Today, approximately 350 million people are chronically infected with the hepatitis B virus and 170 million people are infected with hepatitis C (Marcellin, 2009). Hepatitis C walks in most cases the chronic pathway leading to serious consequences, cirrhosis and hepatocellular carcinoma (Modi & Liang, 2008). According to the World Health Organization, since 1961, chronic hepatitis and cirrhosis have changed from the 10th to the fifth place of death in the United States and Western European countries (Marcellin, 2009).

The use of drugs containing IFN is common for the treatment of leukemias and solid cancers, such as recurrent melanoma (Bukowski et al., 2002; Decantris et al., 2002; Qintas-Cardama et al., 2006).

The effectiveness of drugs containing natural IFNs is limited because of their rapid absorption from subcutaneous tissue, mass distribution, relatively low stability, short half-life, high immunogenicity and toxicity (Wills, 1990). As a result, plasma IFN concentrations were observed to drop rapidly within several hours after administration, and no interferon could be detected in plasma already 24 h after injection (Chatelut et al., 1999). A rapid decrease in interferon concentrations leads to the resumption of viral replication and an increase in viral particle concentrations (Lam et al., 1997). Thus, frequent IFN injections are needed to achieve effective therapeutic concentrations of plasma, which results in dose dependent side effects. This monotherapy of chronic hepatitis C (CHC) with IFN-α2b results in a persistent viral response in only 15-20% of patients for 12 months, with no drug efficacy observed in patients with genotype Ib and high viral load. (McHutchinson et a., 1998).

The therapeutic efficacy of IFNs can be enhanced when using sustained release drugs, especially PEGylated IFNs (Manns et al., 2001; Hadziyannis et al., 2004; Zeuzem et al., 2001). PEGylated IFNs result from chemical conjugation of an interferon molecule with a monomethoxypolyethylene glycol (MPEG) consisting of repeating units of a polymer such as ethylene oxide with a methoxy group at one end and a hydroxy group at the other end. MPEG molecules can have different molecular weights and stereochemical structures (linear or branched). For PEGylation reactions, the hydroxyl groups at the end of MPEG are activated by various reactive functional groups. Activated MPEG can be covalently conjugated with proteins at one or several positions depending on the nature of the activated group and the reaction conditions (Zalipsky & Hurris, 1997; Roberts et al., 2002).

IFN PEGylation may be characterized by excellent pharmacokinetics, increased half-life time, decreased blood levels of fluctuations, decreased immunogenicity and toxicity, increased in vivo activity (decreased in vitro activity); Resulting in increased stability (Glue et al., 2000; Reddy et al., 2001).

The decrease in activity in the in vitro system is dependent on the PEG molecular weight. Conjugation with small PEG molecules with molecular weights up to 5000 Da slightly alters in vitro activity (Grace et al., 2005). The pharmacodynamic and pharmacokinetic properties of PEGylated and unmodified proteins containing this mass of PEG are little different. Conjugation of higher molecular weight PEG results in significant degradation of in vitro biological activity as a result of inefficient conjugation with the receptor, but in this case the pharmacokinetic and pharmacodynamic properties of the PEGylated protein show a significant improvement (Delgado et al. , 1992), which may result in increased biological activity in vivo (Bailon & Berthold, 1998). In addition, PEG structures also affect biological activity and pharmacokinetic properties, and conjugates with linear PEG exhibit larger distribution volumes than conjugates with branched PEG structures (Caliceti et al., 2003).

In addition, the antiviral activity and biological properties of PEG-IFN conjugates depend on the use of other activated MPEG, because of their ability to modify other protein amino acid residues and the type of chemical bonds formed with the protein. Because they are different (Roberts et al., 2002). For protein pegation, activated MPEG, which is capable of conjugation with the free amino groups of the protein, is most widely used (succinimide carbonate, succinimide succinate, trisylate, triazine, hydroxysuccinimide esters, aldehydes). Acetal, etc.).

The following PEG-IFN conjugates are currently known.

PEG-IFN-α2a conjugates with linear PEGs having a molecular weight of 1 to 5000 Da are known (US Pat. No. 5,382,657, 1995). MPEG ethanediol derivatives were used in this conjugation, and the reaction was carried out at room temperature under pH 10 for 0.5 to 4 hours. For the IFN PEGylation reaction, 3-fold excess PEG was used. In the conditions used, the binding of PEG to IFN is achieved through the steric accessibility of the free amino groups of other lysines (Lys-31, Lys133, Lys134, Lys23, Lys131, Lys121, Lys70, Lys83, Lys49 and Lys112). By formation (Monkarsh et al., 1997). Thus, PEG-IFN-α2a consisted of a mixture of positional isomers of the resulting PEG-IFN-α2a conjugate, each of which contained a single PEG. Compared to unmodified IFNs, the specific antiviral activity of the isomers ranged from 6% (Lys112) to 40% (Lys133). The total specific activity of the final conjugate consisting of 11 isomers was 34% of unmodified IFN. In vitro activity of these conjugates was also dependent on the molecular weight of PEG and ranged from 45% (at 2500 Da PEG) to 25% (for 10000 Da PEG). The disadvantages of the final conjugate are as follows:

1. Use of low molecular weight (less than 5000 Da) ethanediol-activated MPEG. As a result, the pharmacokinetic parameters of the PEG-IFN conjugate and the unmodified IFN differed only slightly. As a result, no clinical trials with the conjugates were conducted;

2. Formation of multiple positional isomers with different inactivity due to the conjugation of PEG with IFN molecules via the free amino groups of other lysines.

PEG-IFN-α2b conjugates obtained by conjugation of linear (patent RU 2311930, 2004) or branched (patent RU 2382048, 2008) MPEG derivatives with native IFN-α2b molecular weights of 13000 to 17000 Da are known. The reaction was carried out using 50-100 fold molar excess of activated PEG for 60 hours at pH 9.5 (for linear MPEG) or 12 hours at pH 7.5 (for branched MPEG). As a result, a PEG-IFN conjugate with a total antiviral activity of 29 to 38% of unmodified IFN activity was obtained. There is no data on the conjugate stability, the number of positional isomers and their specific antiviral activity. The disadvantages of these conjugates are as follows:

1. Use of tresylate, a derivative of activated MPEG, with a half-life time of less than 20 minutes in aqueous solution. It has conventionally been found that upon interaction of proteins with tresylate derivatives of PEG, in addition to stable amide bonds via amino groups, unstable sulfamate bonds are also formed (Gais & Ruppert, 1995);

2. The binding of the protein to the tresylate-activated PEG is via all free amino groups of the three-dimensionally accessible lysine (Roberts et al., 2002), where several positional isomers are formed;

3. Performing IFN PEGylation reaction at high pH value (pH = 10) for long time (60 hours) can alter IFN structure;

4. Significant excess of activated PEG (molar ratio of PEG / protein 50 to 100/1) during IFN PEGylation reaction increases the cost of the final product.

There is also a known PEG-IFN-α2b conjugate obtained by conjugation of a branched triazine derivative MPEG having a molecular weight of 7500-35000 Da with native IFN-α2b (RU patent 2298560, 2004), whose total antiviral activity is unmodified. It reached 6.4% of IFN activity. There is no data on conjugation stability, positional isomer number and their antiviral inactivation.

The disadvantages of the obtained conjugates are as follows:

1. Triazines of activated MPEG, which are capable of conjugation with functional groups of other amino acids, serine, tyrosine, threonine and histidine, in addition to the free amino group, can generate many isomers, some of which are characterized by unstable binding. The use of chloride derivatives (Veronese & Pasut, 2005);

2. Low inactivity of PEG-IFN conjugates.

There is also a known PEG-IFN-α2a conjugate obtained by conjugation of a branched PEG having a molecular weight of 40 kDa with IFN-α2a (RU patent 2180595, 1997). For conjugation, an N-hydroxysuccinimide-ester derivative of MPEG was used that selectively interacts with the available free amino groups of the protein to form stable amide bonds. This reaction was carried out for 2 hours at pH 9, 4 ° C. in an MPEG / protein 3: 1 ratio. The reaction was stopped and the resulting conjugate was purified by adsorbent Fractogel EMD CM 650 (M). The yield of purified conjugate was 40-45%. The IFN-PEG conjugate obtained in this case consisted of six positional isomers, each of which was associated with one PEG molecule by stable binding via free amino groups of lysines, Lys31, Lys121, Lys131, Lys134, Lys70 and Lys83. Conjugated (Vailon et al., 2001; Foser et al., 2003). Isomers conjugated with PEG via lysine at positions 31, 121, 131 and 134 were 94% of the total conjugate. The activity of the total conjugate consisting of six positional isomers was 1-7% of native IFN-α2a (Bailon et al., 2001; Boulestin et al., 2006). Despite the low antiviral activity in vitro, the conjugates exhibited improved pharmacokinetic properties compared to unmodified IFNs (Silva et al., 2006; Boulestin et al., 2006) and are currently trademarked "Pegasys" (" Hoffman-La Roche ".

The disadvantages of the obtained conjugates are as follows:

1. The use of N-hydroxysuccinimide-ester-activated PEG allows conjugation with all three-dimensionally accessible amino groups such that the resulting conjugate is a mixture of six isomers with different inactivities.

2. Low in vitro antiviral activity of the obtained conjugate.

The prototype of the present invention is the PEG-IFN-α2b conjugate described in US Pat. No. 5,951,974 (1999). For the PEGylation reaction, linear MPEG (SC-MPEG) activated with succinimide carbonate groups having a molecular weight of 5000 to 12000 Da was used. According to the described method, IFN conjugated with PEG having a molecular weight of 12 kDa was obtained, which is currently sold for the treatment of viral hepatitis under the trade name "PegIntron" ("Schering-Plough", USA).

This PEG-IFN-α2b conjugate ("PegIntron" drug) has been found to consist of 13 positional isomers in which one PEG molecule is conjugated to various parts of the protein molecule, respectively (Wang et al. 2000; Grace et al. , 2001; Youngster et al., 2002). PEG molecules disclosed herein are available through histidine imidazole rings (His7, His34), tyrosine (Tyr129) as well as through free amino lysines (Lys31, Lys49, Lys83, Lys121, Lys131, Lys 133, Lys134, Lys 164) and (Cys1). ) And the serine OH group (Ser163) were also conjugated with interferon. The content of each isomer varied from 0.8% (Tyr129) to 47% (His34). PegIntron's antiviral activity, consisting of 13 positional isomers, is 28% of the native protein. The inactivation of each isomer varied from 11% to 37% of native IFN-α2b (Youngster et al., 2002). The major isomers conjugated with PEG via His34 have the highest inactivation (37% of natural protein). The free amino group of IFN was conjugated to PEG by stable binding, but PEG was conjugated to the histidine imidazole ring (His34) via unstable (in aqueous solution) carbamate linkages in the major isomer of about 47% (Roberts et al., 2002). ).

The pharmacokinetic parameters of this conjugate were superior to unmodified IFNs (Silva et al., 2006).

The disadvantages of the obtained conjugates are as follows:

1. Use of succinimide carbonate activated PEG conjugated not only with the free amino group of IFN but also with the functional groups of other amino acids. As a result, the resulting conjugate consisted of a mixture of 13 positional isomers that differed in antiviral inactivity;

2. Formation of unstable imidazole-carbonate (carbamate) bonds in the major (His-34) isomer of PEG-IFN conjugates results in the release of natural IFNs that undergo hydrolysis to exert biological effects after "Pegintron" administration Brings about. Thus, the drug "PegIntron" was considered the best as a prodrug that hydrolyzes after injection to form active interferon (Foster, 2004). Due to the instability of the PEG-IFN conjugate in solution, the "PegIntron" dosage form is stored only in lyophilized powder form.

It is an object of the present invention that IFN-, consisting of one positional isomer, which improves stability, reduces immunogenicity, improves pharmacokinetic parameters, optimizes the combination of PEG molecular weight and antiviral activity of the conjugate, and is suitable for medical use. It was to obtain stable novel PEGylated IFNs with alpha activity as well as pharmaceutical compositions based on this PEG-IFN conjugate.

The problem is that linear molecules of PEG with interferon-alpha activity and molecular weights from 10000 to 40000 Da are bound with a strictly stable bond to the IFN-α molecule via the alpha amino group of the N-terminal cysteine, It is solved by preparing a novel functionally active PEG-IFN conjugate, which is a compound of:

(I)

Where n is an integer between 227 and 10000;

m-an integer of 4 or more;

IFN-is a natural or recombinant polypeptide with IFN-alpha activity.

In the resulting conjugate, linear PEG with a molecular weight of 10000 to 40000 Da binds to the alpha amino group of the N-terminal amino acid of IFNα.

Stringent selectivity of IFN modification via the alpha-amino group of the N-terminal amino acid is achieved using an aldehyde-activated mPEG of formula (II) via a PEGylation reaction at pH ≤6:

(II)

Wherein n is an integer from 227 to 10000 such that the molecular weight of PEG is from about 10000 to 40000 Da;

m-an integer ≥ 4.

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

Improvements in pharmacokinetic parameters as well as reduction in immunogenicity and toxicity are achieved by increasing the molecular weight of the conjugated PEG.

New features compared to the prototype include:

The novelty compared to the prior art is as follows:

1. An aldehyde derivative of activated mPEG was used for the production of PEG-IFN conjugates;

2. The chemical formula of PEG-IFN conjugate is novel;

3. The produced PEG-IFN conjugate is represented by only one positional isomer;

4. The bond between the PEG molecule and the IFN molecule is stable;

5. The molecular weight of the PEG-IFN conjugate increases due to the size of the bound PEG.

6. Pharmacokinetic parameters have been improved.

For the production of the claimed PEG-IFN conjugates, butyraldehyde or pentaldehyde derivatives of mPEG corresponding to formula (II) having a molecular weight of 20000 Da and recombinant human IFN-α2b (produced by ZAO "Biocad") are used. did.

The conjugation reaction was carried out using a reducing agent at a temperature of 20 ° C. or lower at a pH of 6.0 or lower. PEG / protein molar ratios were between 2.5 and 5: 1. Modulation of PEG-IFN conjugate formation was carried out using polyacrylamide gel electrophoresis (PAGE) and reverse phase high pressure liquid chromatography (RP-HPLC) in the presence of sodium dodecyl sulfate (SDS) under reducing conditions. Purification of monoPEG-IFN from reaction products comprising unmodified IFN and undesirable PEG-IFN forms containing at least two PEG molecules per protein molecule was performed by cation exchange adsorbent chromatography. MonoPEG-IFN elution was performed using a gradient concentration of 0.05-0.2 M of sodium chloride in a buffer of pH 6 or less. Purified monoPEG-IFN products are dialyzed against 10-50 mM buffer (pH 4-5) and are salts, polysaccharides, alcohols, polyvinylpyrrolidone or monosaccharides, and amino sugars, or proteins, or amino acids, and nonionics. Surfactant was added and stored at 4 ± 2 ° C. in plastic or glass bottles with a siliconized surface.

In order to characterize the obtained monoPEG-IFN-α2b conjugate, studies on its purity, uniformity, physicochemical, biological and pharmacokinetic characteristics were performed in comparison with unmodified IFN.

The invention is illustrated by the following figures:

Figure 1. Kinetics of PEG-IFN-α2b conjugate formation during reaction with butyraldehyde MPEG derivatives.
FIG. 2. Reversed phase HPLC analysis on "Symmetry C18" (4.6x150 mm) column of purified PEG-IFN-α2b conjugate.
SDS-PAGE analysis of purified PEG-IFN-α2b conjugates compared to unmodified IFN-α2b.
4. MALDI-MS analysis of unmodified IFN-α2b and PEG-IFN-α2b conjugates.
Figure 5. Location of PEG attachment sites in PEG-IFN-α2b conjugates. Comparison of mass spectra of tryptic peptides in the m / z range 1200-1400 of unmodified IFN-α2b (A) and PEG-IFN-α2b (B) conjugates.
Figure 6. Thermal stability of PEG-IFN-α2b conjugates and unmodified IFN-α2b.
7. Proteolytic stability of PEG-IFN-α2b conjugates and unmodified IFN-α2b.
8. Immunogenicity of PEG-IFN-α2b conjugates compared to unmodified IFN-α2b.
9. Pharmacokinetics of PEG-IFN-α2b conjugates compared to unmodified IFN-α2b.

Specific examples of the PEG-IFN-α2b production method and the results for the study of its properties are shown below.

The PEG-IFN-α2 conjugate of formula (I), which is the object of the present invention, can be used to produce a medicament for the prevention and / or treatment of viral diseases because of its high antiviral activity in addition to its high stability and reduced immunogenicity. In addition, pharmaceutical compositions containing an effective amount of a conjugate of formula (I) produced by known pharmaceutical methods as active ingredients are also infected with viruses (eg, chronically active hepatitis (especially hepatitis B and hepatitis C) (Jay et al. , 2006; McHutchison et al., 2009)), or may be used together or separately with other therapeutic agents (eg, ribavirin) for the prophylaxis and / or treatment. It is also an object of the present invention to provide a method for preventing and / or treating viral diseases such as hepatitis C and hepatitis B, including providing a therapeutically effective amount of a claimed conjugate of formula (I).

IFN-α and PEGylated interferon-α are known to be used as immunomodulators to treat cancer, in particular leukemia retinopathy, laryngeal papilloma, melanoma, renal cell carcinoma, myeloid leukemia, Kaposi's sarcoma (Decatris et al., 2002; Bukowski et al., 2002; Qintas-Cardama et al., 2006; Loquai et al., 2008; Kaehler et al., 2010). Based on the known etiology of these diseases and experience with the use of IFN-α and conjugates for the treatment of these diseases, the object of the present invention is to contain an effective amount of the claimed PEG-IFN-α conjugate and to have antiproliferative and immunomodulatory effects. To provide a drug and pharmaceutical composition that can be used to prevent and / or treat diseases and cancers associated with primary or secondary immunodeficiency, and an object of the present invention is also a PEG-IFN-α conjugate of formula (I) To provide a method of preventing and / or treating diseases and cancers associated with primary or secondary immunodeficiency, including providing a therapeutically effective amount of.

The conjugate of formula (I) may optionally be administered intravenously, subcutaneously, intramuscularly or any other suitable route, such as capsules, syrups, sprays, together with pharmaceutically acceptable fillers, diluents and / or pharmaceutically acceptable excipients, It is administered in the form of drops, injections or suppositories. The route of administration depends, for example, on the symptoms and age. Frequency of administration and interval of injection depend on the disease and its severity or purpose of administration (treatment or prophylaxis). The effective amount of PEG-IFN-α is selected according to the factors mentioned above.

Pharmaceutically acceptable excipients may be included in the pharmaceutical composition with PEG-IFN-α: buffer salts (eg acetate, citrate, hydrocarbonate, phosphate buffer), stabilizers (eg polysorbate, EDTA, polyvinyl pi) Lollidon, dextran, human serum albumin, ether paraoxybenzoic acid, alcohol (e.g. benzyl alcohol), phenol, sorbic acid), preservative component (e.g. benzyl alcohol), osmotic pressure regulator (e.g. sodium chloride, potassium chloride, polyol, e.g. Glycerol, arabitol, sorbitol, mannitol, lactose, dextran), surfactants (e.g. HSA, polyvinylpyrrolidone, lecithin, polysorbate 80, polyoxyethylene-polyoxypropylene copolymer, casein), surface activity Substances (e.g. ethylene oxide and propylene oxide, propylene oxide and ethylene oxide, sorbitan monolaurate, sorbitol esters, polyglycerol fatty acid esters , Cocamide DEA lauryl sulfate, alkanolamide, stearyl polyoxyethylene propylene glycol, lauric ether polyoxyethylene, polyoxyethylene cetyl ether, polysorbate, glycerol monostearate, glycerol distearate, sorbitol monopalmi Tate, sorbitan monooleate polyoxyethylene, sorbitan monolaurate polyoxyethylene monostearate and propylene glycol), antioxidants (e.g. Trilon B, L-cysteine, sodium sulfite, sodium ascorbate, glutathione, 2- Mercaptoethanol, dithiothreitol, cysteine hydrochloride monohydrate, ascorbic acid) and diluents (eg water).

Example  One

Butyraldehyde MPEG  Derivatives PEG - IFN Production of -α2b Conjugates

16 ml of 1 M sodium cyanoborohydride solution was added to 785 ml of buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.0) containing recombinant IFN-α2 at a concentration of 1.7 mg per ml, and then the solution was stirred and molecular weight 5 g of this anhydrous butyraldehyde PEG derivative which was 20000 Dalton were added. The mixture was stirred sufficiently and incubated for 22 hours at a temperature of 17 ± 3 ° C. 50 μl aliquots were taken from the mixture after several time intervals and the production kinetics of PEG-IFN-α2 conjugates were analyzed by polyacrylamide electrophoresis in the presence of dodecyl sulfate. For this purpose, buffer (pH 6.8) containing 125 mM Tris-HCl, 20% glycerin, 3% dodecyl sulfate, 0.005% bromphenol blue is added to the mixture in an amount of 1/3 of the total volume, and in a boiling bath Heated for 3 min. 5 μl of the sample was placed in a well of the prepared 12.5% polyacrylamide gel slab and electrophoresis was performed in the presence of dodecyl sulfate. After completion of electrophoresis, the gels were stained with Coomassie R-250. Production kinetics of PEG-IFN-α2 conjugates are shown in FIG. 1. At a PEG-IFN content of at least 70%, the reaction mixture was diluted 10-fold with 5 mM sodium acetate buffer (pH 5.0).

Example  2

Pentaldehyde MPEG  Derivatives PEG - IFN Production of -α2b Conjugates

2.05 ml of 1M sodium cyanoborohydride solution was added to 100 ml of a buffer solution (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5) containing recombinant IFN-α2b at a concentration of 2.2 mg per ml, followed by stirring; 0.9 g of anhydrous pentalaldehyde PEG derivative having a molecular weight of 20000 Da was added. The mixture was stirred sufficiently and incubated for 24 hours at a temperature of 6 ± 2 ° C. 50 μl aliquots were taken from the mixture after several time intervals and the production kinetics of the PEG-IFN conjugates were analyzed using the polyacrylamide gel electrophoresis described in Example 1. At a PEG-IFN content of at least 70%, the reaction mixture was diluted 10-fold with 5 mM sodium acetate buffer (pH 5.0).

Example  3

Mono by ion exchange chromatography in cation exchange adsorbents PEG - IFN Purification of -α2b Conjugates

The dilution solution produced in Example 1 was loaded at a flow rate of 5 ml / min to a column filled with a cation-exchange adsorbent (CM-Sepharose, 300 ml) equilibrated with 5 mM sodium acetate buffer pH 5.0 (buffer A). The adsorbent column was washed successively with Buffer A and Buffer A containing a NaCl concentration gradient of 0.05M to 0.2M. Aliquots were taken from each fraction for the purpose of analyzing the samples by electrophoresis on polyacrylamide gels as described in Example 1. Fractions containing monoPEGylated PEG-IFN-α2 conjugates were combined, dialyzed with 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, followed by sterile filtration and the resulting solution stored at 4 ± 2 ° C.

Example  4

Mono by ion exchange chromatography in cation exchange adsorbents PEG - IFN Purification of -α2b Conjugates

The dilution solution produced in Example 2 was loaded at a flow rate of 5 ml / min into a column filled with a cation-exchange adsorbent (SP Sepharose, 50 ml) equilibrated with 5 mM sodium acetate buffer pH 5.0 (buffer A). The adsorbent column was washed successively with Buffer A and Buffer A containing a NaCl concentration gradient of 0.03 M to 0.3 M. Aliquots were taken from each fraction for the purpose of analyzing the samples by electrophoresis on polyacrylamide gels as described in Example 1. Fractions containing monoPEGylated PEG-IFN-α2 conjugates were combined, dialyzed with 10 volumes of 20 mM sodium acetate buffer containing 150 mM sodium chloride, followed by sterile filtration and the resulting solution stored at 4 ± 2 ° C.

Example  5

PEG - IFN  Conjugate Inverse phase - HPLC  analysis

The PEG-IFN-α2 conjugate produced in Example 3 was diluted with 20 mM sodium acetate buffer pH 5.0 to a minimum concentration of 0.1 mg / ml per ml, and 100 μl of this sample was loaded onto a Symmetry C18 column (4.6 × 150 mm). The analysis was performed at 214 nm using "Breeze" chromatography from Waters Company. Based on the results presented in FIG. 2, it can be concluded that the purity of the claimed PEG-IFN-α2 conjugate according to the RP-HPLC results is greater than 99%.

Example  6

Endotoxin Level Measurement

The concentration of bacterial endotoxin (BE) in the PEG-IFN-α2 conjugate samples produced according to Examples 3 and 4 was measured in vitro using the LAL test (gel coagulation version) according to the requirements of Section 2.6.14 of the European Pharmacopoeia 6.0. did. This analysis includes the diagnostic kit Associates of CAPE COD, Inc. Product, LAL reagent (sensitivity 0.03 EU / ml), endotoxin reference standard (0.5 μg in Bayer), and water for LAL testing were used. Based on the results presented in Table 1, it can be concluded that the BE content in the conjugate samples is less than 1.5EU (mg of endotoxin units) per mg of protein, which is much lower than the level of BE that is acceptable for drugs based on recombinant proteins. have.

Bacterial Endotoxin Concentration in PEG-IFN-α2 Conjugates PEG-IFN-α2 Conjugate Formulations Endotoxin Content (EU / mg Protein) PEG-IFN-α2 prepared according to Example 3 ≤1.5 PEG-IFN-α2 prepared according to Example 4 ≤1.4

Example  7

Undeformed IFN -α2b contrast PEG - IFN Electrophoretic Analysis of -α2b Conjugates

PEG-IFN conjugate samples prepared according to Example 3 were analyzed by polyacrylamide gel electrophoresis as described in Example 1. Electrophoresis was performed with a loading of 40 μg protein per well under non-reducing conditions. Protein staining of the gel was performed with Coomassie R-250 dye. At the same time, electrophoresis of unmodified IFN-α2 was also performed. PEG-IFN conjugates appeared in a single band (FIG. 3, track 1). Based on the electrophoretic curves of the PEG-IFN-α2 conjugate and the unmodified IFN-α2 shown in FIG. 3, it can be seen that the PEG-IFN-α conjugate has a higher molecular weight than the unmodified IFN-α (FIG. 3, Track 1 and track 2). It should be noted that the exact molecular weight values of PEGylated proteins are impossible to measure by electrophoresis because the addition of hydrophilic PEG molecules to the protein increases the Stokes radius of the final compound. As a result, the migration of PEG-protein compounds in the gel is slowed down, so that the molecular weight appears to be much higher than the sum of the molecular weights of the protein and PEG.

Example  8

Unstrained by Mass Spectrometry IFN compared to α2b, PEG - IFN  Molecular Weight Measurement of α2b Conjugates

1 μl PEG-IFN-α2b conjugate sample prepared according to Example 3 and 2,5-dihydroxybenzoic acid solution (Aldrich, 10 mg × ml ⁻¹ in 20% acetonitrile solution containing 0.5% uranium tetrafluoride) 0.3 μl was mixed and dried in air. In the same manner, an unmodified IFN-α2 sample was prepared.

Mass spectra were obtained on an Ultraflex II BRUKER (Germany) MALDI-TOF mass spectrometer equipped with a UV laser (Nd). Mass spectra were obtained in positive ion mode; The average gravimetric error does not exceed 10-15 Da.

Mass spectrometric results of PEG-IFN-α2 conjugates and unmodified rhIFN-α2 in the range m / z 10000 to 50000 are shown in FIG. 4. Based on the results presented in FIG. 4A, it can be concluded that the mass spectrum of rhIFN-α2 contains a base peak corresponding to [M] + monovalent ions having a molecular weight of 19295 Daltons.

In the mass spectrum of the PEG-IFN-α2 conjugate shown in Figure 4B we can see the diffused main peak corresponding to [M] + monovalent ions having a molecular weight of 40498 Da. The spread of the peak is due to the heterogeneity of the monomethoxyPEG preparation. The measured m / z value of the PEG-IFN conjugate at the maximum peak (40498 Da) is consistent with the calculated sum of the molecular weights of IFN-α2 (19295 Da) and PEG (20000 Da) attached.

Example  9

PEG - IFN in -α2b conjugates PEG Angry Locality

5 μl of a modified trypsin (Promega) solution dissolved in 0.05M NH ₄ HCO _{3 at a} concentration of 15 μg × ml ⁻¹ was added to 5 μl of a PEG-IFN conjugate sample prepared according to Example 3. Hydrolytic digestion was carried out at temperature 37 ° C. for 16 h, then 10 μl of a 0.5% uranium tetrafluoride solution in 10% acetonitrile aqueous solution was added and stirred well. Unmodified IFN samples were prepared in a similar manner. The final solution was used to obtain MALDI mass spectra.

Localization of the IFN molecule and PEG binding site was determined by comparing the mass spectra of the PEG-IFN-α2 conjugate and the unmodified IFN-α2 after trypsin digestion. PEG-IFN-α2 conjugate trypsinates should be free of peptides corresponding to the portion of the protein that has been modified. In Table 2 the mass spectra of the experimental trypsinates of the unmodified IFN-α and PEG-IFN-α2 conjugates can be seen. Based on the results presented in this table, it can be concluded that the mass spectrum of the trypsinate of the unmodified IFN-α2b substantially matches the mass spectrum of the trypsinate of the PEG-IFN-α2 conjugate (Table 2). The peptide of molecular weight 1313.649 present in the trypsin digest of the modified IFN-α2 is not present in the trypsin digest of the PEG-IFN-α2 conjugate (see FIG. 5). According to the theoretical weight analysis of IFN-α2 trypsin digest, the peak at m / z 1313.649 corresponds to the N-terminal protein peptide (Table 2). The absence of this peptide in the trypsin digest of the PEG-IFN-α2 conjugate demonstrates the modification of the N-terminal peptide and is heavier due to the weight of the PEG, outside the region of the investigated spectrum. PEG binding to the IFN molecule occurred only on the N-terminal cysteine amino group, which is the only free amino group present on this peptide.

Experimental mass spectra of trypsinates of PEG-IFN-2 conjugates and unmodified IFN-2 compared to theoretical values Experimentally Observed Peptides
[M + H ⁺ ] ⁺ Theoretical peptide IFN-a2b PEG-IFN Conjugate Mass [M + H ⁺ ] ⁺ Peptides order 1313.649 - 1313.6266 1-12 CDLPQTHSLGSR 1232.720 1232.673 1232.6966 13-22 RTLMLLAQMR 1076.598 1076.558 1076.5955 14-22 TLMLLAQMR 910.463 910.463 910.5066 24-31 ISLFSCLK 2225.976 2225.928 2225.9999 32-49 DRHDFGFPQEEFGNQ 2459.264 2459.239 2459.3002 50-70 AETIPVLHEMIQQIFN 772.447 772.421 772.456 99-106 VIQGVGVT 902.471 902.443 902.4941 113-120 EDSILAVR 1030.590 1030.546 1030.5891 113-121 EDSILAVRK 741.431 741.424 741.4042 121-125 KYFQR 750.492 750.476 750.4760 126-131 ITLYLK 1337.695 1337.651 1337.6670 134-144 KYSPCAWEVVR 1209.565 1209.539 1209.5721 135-144 YSPCAWEVVR 1481.783 1481.735 1481.7594 150-162 SFSLSTNLQESLR

Example  10

PEG - IFN Measurement of Antiviral Inactivation of -α2b Conjugates

Samples prepared according to Examples 3 and 4 were subjected to the method described in the European Pharmacopeia using subcultured MBDK cell cultures and bullous stomatitis virus (VSV) cultures that are sensitive to alpha-type interferon for antiviral inactivation. Tested accordingly. Table 3 presents the results of the study of antiviral activity. International reference standards were used at the reference level. From the results in Table 3, it can be concluded that despite the conjugation with PEG molecules having a molecular weight of 20000 Da, the antiviral activity of the produced conjugates is 42-45% of the unmodified IFN-α2. It should be noted that the PEG-IFN conjugates described in the prototype method containing PEG with a molecular weight of 12000 Da have lower activity (28-30%).

Antiviral Inactivation of PEG-IFN-α2 Conjugates Against Unmodified IFN-α2 Product Antiviral inactivity (IU per mg) % Of unmodified IFN-α2b activity IFN-α2 2.1 x ^{10 8} 100% PEG-IFN-α2 Conjugates Prepared According to Example 3 0.7x10 ⁸ 40% PEG-IFN-α2 Conjugates Prepared According to Example 4 0.9 x ^{10 8} 45%

Example  11

PEG - IFN of -α2b conjugates Thermal stability  Research

5 ml of the PEG-IFN conjugate sample prepared according to Example 3 was placed in a water bath at a temperature of (50 ± 2) ° C., and after another time interval, turbidity of the protein solution was examined by optical density measurement at 340 nm. In a similar manner, thermal stability studies of unmodified IFN-α2 were performed. Formation of insoluble protein compounds resulted in turbidity and increased optical density at 340 nm. The results of this study are presented in FIG. 6. During the observation period (28 hours), the PEG-IFN-α2 conjugate remained stable while the unmodified IFN-α2b precipitated almost completely after 28 h.

Thus, the data obtained in this study leads to the conclusion that the thermal stability of PEG-IFN-α2 conjugates is substantially increased compared to unmodified proteins.

Example  12

When trypsin treatment, PEG - IFN Proteolytic Stability Studies of -α2b Conjugates

To 1 ml PEG-IFN conjugate prepared according to Example 3, 15 µl of 20 µg trypsin (Promega), ₂ µl of 0.5 M CaCl ₂ solution and 150 µl of 1 M Tris-HCl buffer (pH 8.5) were added per ml. The sample was incubated at a temperature of 37 ° C. An aliquot of 100 μl was taken from this mixture after another time interval, and the reaction was stopped by adding 150 μl of trifluoroacetic acid solution. Samples containing unmodified IFN-α2 were prepared in a similar manner. The sample was then analyzed by reverse phase HPLC, measuring the area of the main peak. The study results presented in FIG. 7 demonstrate that upon trypsin treatment, the proteolytic stability of PEG-IFN-α2 conjugates is higher than unmodified IFN-α2b.

Example  13

PEG - IFN Storage Stability Measurement of -α2b Conjugates

An aliquot was taken from the sample prepared according to Example 3 and injected into an Eppendorf test tube with a cap. The protein concentration of PEG-IFN-α2 conjugate was 1 mg per ml. This sample was stored in a refrigerator at (6 ± 2) ° C. Samples were taken after a predetermined time interval and analyzed by polyacrylamide gel electrophoresis (Eph) in the presence of dodecyl sulfate at RP-HPLC and non-reducing conditions described in Example 1 for antiviral inactivation and uniformity of the formulation. Based on the results presented in Table 4, it can be concluded that PEG-IFN-α2 conjugates are stable for at least 24 months during storage at (6 ± 2) ° C.

PEG-IFN-α2 conjugate stability at a temperature of (6 ± 2) ° C parameter
Shelf life (month) 0 3 6 9 12 18 24 activation
(IU / ml) 0.7 × 10 ⁸ 0.75 × 10 ⁸ 0.7 × 10 ⁸ 0.75 × 10 ⁸ 0.72 × 10 ⁸ 0.7 × 10 ⁸ 0.75 × 10 ⁸ Uniformity,%
(RP-HPLC) 99.67 99.28 99.61 99.30 99.6 99.21 99.35 Uniformity,%
(SDS-PAGE) ≥98 ≥98 ≥98 ≥98 ≥98 ≥98 ≥98

Example  14

PEG - IFN Immunogenicity Studies of -α2b Conjugates

5 mln of the PEG-IFN conjugate prepared according to Example 3 and Example 4. Diluted at a concentration of IU / ml, ICR mice weighing 20-22 g were injected intramuscularly once weekly for 5 weeks at 200 μl dose (1 mln IU / mouse) (5 groups of 5 mice per group). At the same time, unmodified IFN samples were similarly prepared and injected into mice at a 1 mln.IU dose. Blood samples were taken from the mice at the last 5 weeks after puncture. Blood serum was received by standard methods. Antibody titers in serum obtained after mouse immunization with unmodified IFN and PEG-IFN conjugates were measured by direct ELISA technique. To this end, 100 μl of unmodified IFN-α2 solution at a concentration of 200 ng per 100 μl was injected into the microplate wells. Antisera obtained from different groups of animals was continuously injected into wells of the plate from dilution serially diluted in two replicates. To detect the final immune complexes, anti mouse antibodies conjugated with peroxidase were used. Antibody titer after administration of unmodified IFN was 1/1024. The antibody titer after administration of the PEG-IFN conjugate was 1/128. The results of this study are presented in FIG. 8.

Thus, based on the results obtained, it can be concluded that the immunogenicity of the claimed PEG-IFN-α2 conjugate is 8 times lower than unmodified IFN.

Example  15

PEG - IFN Pharmacokinetic Study of -α2b Conjugates

A sample of 1 mln IU amount of PEG-IFN conjugate prepared according to Example 3 was injected intraperitoneally into male mice. Side by side, mice in other groups were injected with unmodified IFN-α2. Animal blood samples were taken by a posterior eye puncture after a predetermined time interval. Blood serum was obtained by standard methods. The presence of interferon in serum was detected based on antiviral activity. The pharmacokinetic study results are presented in FIG. 9. Based on these results, it can be concluded that the claimed PEG-IFN-α2 conjugates have a much longer effect. After native IFN-α2 injection, its animal blood concentration reached a maximum 1 h after injection and then decreased very rapidly. After injection of the PEG-IFN conjugate, the maximum IFN concentration in the animal blood was observed after 12 hours (FIG. 9), this level was preserved for 50 hours, after which a slow decrease in IFN-α2 concentration was observed for 350 hours.

The area under the curve (AUC) of the claimed PEG-IFN conjugates was 50 times greater than each value of unmodified IFN. Note that for circular PEG-IFN-α2b conjugates coupled with PEG with a molecular weight of 12000 Da, the AUC values exceeded IFN AUC values only 3 to 10 times.

Based on the results obtained (FIG. 9), the main pharmacokinetic parameters of the claimed PEG-IFN-α2 conjugates were calculated. This result shows that absorption from the injection site, distribution dose, and removal rate of PEG-IFN conjugates are significantly slower than unmodified IFNs, which allows the claimed PEG-IFN conjugates to circulate in the blood for a long time (more than 14 days). Showed that

Example  16

Charged PEG - IFN -α2b conjugates and primitives ( prototype Comparison of the properties of PEG-IFN-α2b conjugates as described in the method

A comparison of the structure, basic physicochemical parameters and pharmacokinetic parameters of the claimed PEG-IFN-α2b conjugates was performed as compared to the PEG-IFN-α2b conjugates described in the prototype method (Table 5). The data presented in Table 5 demonstrate that the properties of the claimed PEG-IFN conjugates were significantly improved over the conjugates described in the prototype method.

Basic physicochemical and pharmacokinetic parameters of the claimed PEG-IFN-α2b conjugate compared to the PEG-IFN-α2b conjugate described in the original method (US Pat. No. 5,951,974) parameter
PEG-G-CSF Conjugate Claimed PEG-G-CSF Conjugates Conjugates Described in Circular Method (US Patent 5,951,974) IFN IFN-α2b IFN-α2b PEG structure Linear Linear PEG molecular weight (kDa) 20 12 PEG-IFN Conjugate Structural Formula *

% Of inactive, natural IFN 40 28 Number of positional isomers One 13 PEG binding type for proteins Stable Unstable (major isomers bind to PEG via His34) Stability in aqueous solution Stable Instability Pharmacokinetic parameters AUC ratio of PEG-IFN / IFN 50 3-10

* PEG-IFN conjugate structural formula obtained by the circular method set forth in the book "WHO Drug Information", 2001, v. 15, N.3-4, p.206.

Example  17

Charged PEG - IFN Examples of pharmaceutical compositions based on -α2b conjugates

Example  18

PEG - IFN Packaging of Drug Formulations Containing -α2b

Drug formulations containing an effective amount of the claimed PEG-IFN-α2b (Example 17) have doses of 0.5, 0.8, 1.0 and 1.2 ml (when 100 μg per 1 ml dose), 0.5, 0.6, 0.75 and 0.9 ml ( Elastomeric or rigid, sealed with a nozzle on a butyl rubber plunger, laminated with a fluoropolymer of 200 μg per 1 ml dose), 0.5, 0.6, 0.8, 1.0 and 1.2 ml (300 μg per 1 ml dose). Dispensed under sterile conditions in a syringe made of hydrolyzable Class 1 neutral glass with a brass-inscribed needle with a protective cap.

Example  19

PEG - IFN Packaging of Drug Formulations Containing -α2b

Drug formulations containing an effective amount of PEG-interferon alpha-2b (Example 17) were 0.5, 0.8, 1.0 and 1.2 ml (when 100 μg per 1 ml dose), 0.5, 0.6, 0.75 and 0.9 ml (1 ml dose) 200 μg per sugar), 0.5, 0.6, 0.8, 1.0 and 1.2 ml (300 μg per 1 ml dose), sealed with a nozzle or a fluorinated rubber on a Teflon-coated butyl rubber cover tightened with an aluminum cap However, the vials made of hydrolyzable class 1 neutral glass should be dispensed under sterile conditions.

Example  20

PEG - IFN comprising a packaged drug formulation containing -α2b Kit

The kit contains a plunger (correspondingly one or four) with a Bayer or one or four syringes in a blister made of a polymer film with the treatment information placed in a board pack.

references

RU patent 2311930, 2004;

RU patent 2382048, 2008;

RU patent 2298560, 2004;

RU patent 2180595, 2005;

U.S. Patent 5,951,974, 1999;

Claims (25)

Stable PEGylated interferon-α conjugates represented by formula (I) and consisting of one positional isomer:
(I)

[Wherein n is an integer from 227 to 10000; m is an integer of ≧ 4; N ^α H-IFN is interferon-α]. 2. The conjugate of claim 1, wherein m is an integer of 4. The conjugate of claim 1, wherein the interferon-α is natural or recombinant interferon-α-2b. 2. The conjugate of claim 1 wherein the polyethylene glycol has an average molecular weight of 10 to 40 kDa. The conjugate of claim 1, wherein the N-terminal group is a cysteine residue (Cys). A pharmaceutical composition having antiviral, antiproliferative and immunomodulatory activity, containing an effective amount of the conjugate of formula (I) and a pharmaceutically acceptable excipient. 7. A pharmaceutical composition according to claim 6 for use as a drug for the treatment of diseases associated with primary or secondary immunodeficiency and viral and oncological diseases. 8. The pharmaceutical composition of claim 7, wherein the viral disease is hepatitis C or hepatitis B. 8. The pharmaceutical composition of claim 7, wherein the oncological disease is myeloid leukemia. 8. The pharmaceutical composition of claim 7, wherein the oncological disease is melanoma. A pharmaceutical preparation with antiviral, antiproliferative and immunomodulatory activity, comprising a conjugate of formula (I). The drug formulation according to claim 11, which contains a buffer, an isotonic agent, a stabilizer and water. 13. A drug formulation according to claim 12 containing sodium acetate trihydrate, acetic acid, sodium chloride, polysorbate 80, EDTA, water for injection. Use of a conjugate represented by formula (I) for use in the production of drug formulations with antiviral, antiproliferative and immunomodulatory activity. Use according to claim 14 for the production of a drug formulation with antiviral activity against hepatitis C or hepatitis B. A method for preventing and / or treating a viral disease comprising providing a therapeutically effective amount of a conjugate represented by formula (I). The use according to claim 16, wherein the viral disease is hepatitis C or hepatitis B. The use of claim 16 further comprising providing a therapeutically effective amount of ribavirin. A method for preventing and / or treating a disease associated with primary or secondary immunodeficiency, comprising providing a therapeutically effective amount of a conjugate represented by formula (I). A method for preventing and / or treating an oncological disease comprising providing a therapeutically effective amount of a conjugate represented by formula (I). The use of claim 20, wherein the oncological disease is myeloid leukemia. The use of claim 20, wherein the oncological disease is melanoma. A hermetically sealed and sterile hermetically sealed container comprising the pharmaceutical composition according to claim 6. The container of claim 23, wherein the particular container is a prefilled syringe, bayer or autoinjector. A kit comprising the container and treatment information of claim 24.

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