WO2012011836A1 - The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer - Google Patents

The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer Download PDF

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WO2012011836A1
WO2012011836A1 PCT/RU2010/000529 RU2010000529W WO2012011836A1 WO 2012011836 A1 WO2012011836 A1 WO 2012011836A1 RU 2010000529 W RU2010000529 W RU 2010000529W WO 2012011836 A1 WO2012011836 A1 WO 2012011836A1
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conjugate
ifn
peg
formula
interferon
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French (fr)
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Dmitriy Valentinovich Morozov
Tatyana Veniaminovna Chernovskaya
Lev Alexandrovich Denisov
Elena Georgievna Rudenko
Angelina Vsevolodovna Klenova
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Closed Joint Stock Company "Biocad"
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Priority to KR1020137001858A priority Critical patent/KR101586372B1/ko
Priority to SG2013003801A priority patent/SG187117A1/en
Priority to CUP2013000013A priority patent/CU24193B1/es
Publication of WO2012011836A1 publication Critical patent/WO2012011836A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention is related to pharmaceuticals, namely, to physiologically active conjugates of interferon, in particular, to new conjugates of interferon with polyethylene glycol (PEG), that can be used in medicine, for example, for treatment of viral, immunological and oncological diseases.
  • PEG polyethylene glycol
  • Interferons are a group of biologically active proteins or glycoproteins that are produced by various cells in response to viral infection or as a result of impact of certain chemical and biological substances on cells (Isaacs & Lindeman, 1957; Pestka et al., 2007). Conjugation of IFN with cell receptors leads to induction of a number of intracellular proteins that mediate antiviral, immunomodulatory and antiproliferative IFN effects (Pestka et al., 2004; Bekisz, 2004).
  • PEGylated IFNs result from chemical conjugation of interferon molecule with the polymer - for example, monomethoxypolyethylene glycol (MPEG), consisting of repeating residues of ethylene oxide with a methoxy group at one terminus and a hydroxyl group - at another.
  • MPEG molecule may have different molecular weight and the stereochemical structure (linear or branched).
  • hydroxyl group at the end of MPEG is activated by various reactive functional groups.
  • Activated MPEG can be covalently conjugated with a protein in one or several locations, depending on the nature of the activated group and the reaction conditions (Zalipsky & Hurris, 1997; Roberts et al, 2002).
  • IFN PEGylation leads to better pharmacokinetics, increased half-life time, reduced fluctuations of concentration in the blood, reduced immunogenicity and toxicity, increased activity in vivo (with decrease in activity in vitro); increased stability (Glue et al, 2000; Reddy et al, 2001).
  • PEG structure also affects the biological activity and pharmacokinetic properties - conjugate with linear PEG has a larger distribution volume than conjugate with branched PEG structure (Caliceti et al, 2003).
  • Antiviral activity and biological properties of PEG-IFN conjugates depend also on the usage of different activated MPEGs, since their functional groups vary by their ability to modify different protein amino acid residues and by the type of chemical bonds formed with the protein (Roberts et al, 2002).
  • activated MPEGs that are able to conjugate with free amino groups of proteins are most widely used (succinimide carbonate, succinimide succinate, trisilate, triazine, hydroxysuccinimide esters, aldehyde acetal, etc.).
  • a PEG-IFN-a2a conjugate with linear PEG with molecular weight 1-5 000 Da is known (Patent US No. 5,382,657, 1995).
  • IFN PEGylation reaction 3 -fold excess of PEG was used.
  • PEG-IFN-oc2a consisted of a mixture of the resulting positional isomers of PEG-IFN-a2a conjugate, where each isomer contained a single PEG. In comparison with the unmodified IFN specific antiviral activity of isomers ranged from 6% (for Lysl 12) to 40% (for Lys 133).
  • the disadvantages of the resulting conjugate are as follows:
  • PEG-IFN-a2b conjugates obtained by conjugation of native IFN-a2b with linear (patent RU 231 1930, 2004) or branched (patent RU 2382048, 2008) MPEG derivatives with molecular weight 13 000 - 17 000 Da.
  • the reaction was conducted at pH 9.5 for 60 hours (for the linear MPEG) or at pH 7.5 for 12 hours (for the branched MPEG), using a 50- 100-fold molar excess of activated PEG.
  • PEG-IFN conjugates have been obtained, the total antiviral activity of which varied from 29 to 38% of the activity of unmodified IFN. Data about conjugate stability, the number of positional isomers and their specific antiviral activity are absent.
  • the disadvantages of these conjugates are as follows:
  • PEG-IFN-cc2b conjugate obtained by conjugation of native IFN-a2b with branched triazine derivative MPEG with molecular weight 7500 - 35 000 Da (Patent RU 2298560, 2004), the total antiviral activity of which amounted to 6,4% of the activity of unmodified IFN. Data about conjugation stability, the number of positional isomers and their specific antiviral activity are absent.
  • triazine-chloride derivatives of activated MPEG which are able to conjugate with functional groups of other amino acids - serine, tyrosine, threonine, and histidine in addition to free amino groups, which leads to the emergence of many isomers, some of which are characterized by an unstable bond.
  • triazine derivatives are currently not used because of their high toxicity (Veronese & Pasut, 2005);
  • PEG-IFN-a2a conjugate obtained by conjugation of IFN-a2a with branched PEG with molecular weight 40 kDa (patent RU 2180595, 1997).
  • a N- hydroxysuccinimide-ester derivative of MPEG was used, which selectively interacts with the available free amino groups of protein to form stable amide bond.
  • the reaction was conducted at pH 9,4°C for 2 hours at the 3: 1 ratio of MPEG / protein.
  • the reaction has been stopped and obtained conjugate has been purified by the sorbent Fractogel EMD CM 650 (M). Yield of the purified conjugate was 40-45%.
  • the obtained IFN-PEG conjugate consisted of six positional isomers, each of which was conjugated with one PEG molecule by a stable bond through free amino groups of lysines - Lys31, Lysl21, Lys 131, Lys 134, Lys 70 and Lys 83 (Vailon et al, 2001 ; Foser et al, 2003). Isomers conjugated with PEG through lysines in positions 31, 121, 131 and 134 formed 94% of the total conjugate. The activity of the total conjugate consisting of 6 positional isomers, was 1-7% of the native IFN-a2a (Bailon et al, 2001 ; Boulestin et al, 2006).
  • N-hydroxysuccinimide-ester activated PEG leads to conjugation with all sterically accessible amino groups, as a result the obtained conjugate is a mixture of 6 isomers, differing in specific activity.
  • the prototype of this invention is the PEG-IFN-cc2b conjugate described in the patent US 5,951,974, 1999.
  • a linear MPEG activated by succinimide carbonate group (SC-MPEG) with molecular weight from 5 000 to 12 000 Da was used.
  • SC-MPEG succinimide carbonate group
  • an IFN conjugated with PEG with molecular mass of 12 kDa was obtained, which is currently on the market for treatment of viral hepatitis under the trade name "Peglntron” ("Schering-Plough", USA).
  • PEG-IFN-a2b conjugate (medication "Peglntron”) consists of 13 position isomers, each of which contains one PEG molecule conjugated with various parts of the protein molecule (Wang et al. 2000; Grace et al., 2001 ; Youngster et al, 2002).
  • PEG molecule was conjugated with interferon not only through free amino lysines (Lys31 , Lys 49, Lys 83, Lys 121, Lys 131, Lys 133, Lys 134 H Lys 164) and (Cysl), but also through histidine imidazole ring (His7, His34), tyrosine (Tyrl29) and serine OH-group (Serl63). Content of individual isomers varied from 0,8 (Tyrl29) to 47 % (His34). Antiviral activity of Peglntron consisting of 13 position isomers is 28 % of the native protein.
  • Specific activity of individual isomers varied from 1 1 to 37 % of the native IFN-oc2b (Youngster et al, 2002).
  • the main isomer conjugated with PEG through His34 has the highest specific activity (37 % of the native protein).
  • the free amino group of IFN has been conjugated with PEG by a stable bond, while in the main isomer, comprising about 47%, PEG was conjugated with the histidine imidazole ring (His34) through the unstable (in aqueous solutions) carbamate bond (Roberts et al., 2002).
  • the disadvantages of the obtained conjugate are the following: 1.
  • obtained conjugate consisted of a mixture of 13 positional isomers, differing in antiviral specific activity;
  • the aim of the present invention was to obtain a new stable PEGylated IFN with the activity of IFN-alpha, consisting of one positional isomer, with improved stability, with reduced immunogenicity, improved pharmacokinetic parameters, with the optimal combination of PEG molecular weight and antiviral activity of the conjugate, suitable for medical use, as well as pharmaceutical compositions based on these PEG-IFN conjugate.
  • n - integral values from 227 to 10 000;
  • IFN - natural or recombinant polypeptide having the activity of IFN-alpha having the activity of IFN-alpha.
  • conjugate linear PEG with the molecular weight 10 000 - 40 000 Da is bound with the alpha amino group of the N-terminus amino acid of IFNa.
  • the conjugation reaction was performed at pH below 6.0 with a reducing agent at a temperature at or below 20 °C.
  • the molar ratio of PEG / protein was 2,5-5 : 1.
  • Control of PEG- IFN conjugate formation was performed using polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) in reducing conditions and reversed-phase high pressure liquid chromatography (RP-HPLC). Purification of monoPEG-IFN from the reaction products that included unmodified IFN and undesirable forms of PEG-IFN, containing two or more PEG molecules per protein molecule, was carried out by chromatography on cation- exchange sorbents.
  • MonoPEG-IFN elution was performed by gradient concentration of sodium chloride from 0,05 to 0,2 M in buffer solution with a pH below 6.
  • the purified monoPEG-IFN product was dialyzed against 10-50 mM buffer solution at pH 4-5, adding salt, or polysaccharides, or alcohols, or polyvinylpyrrolidone, or monosaccharides, and amino sugars, or proteins, or amino acids, and nonionic detergents and stored in plastic or glass bottles with siliconized surface at the temperature of 4 ⁇ 2 °C.
  • Fig. 3 SDS-PAGE analysis of the purified PEG-IFN-cc2b conjugate compared to unmodified IFN-a2b.
  • Fig. 4 MALDI-MS analysis of unmodified IFN-a2b and PEG-IFN-a2b conjugate.
  • Fig. 5 Location of PEG attachment site in the PEG-IFN-ct2b conjugate. Comparison of mass spectra of tryptic peptides of unmodified IFN-a2b (A) and PEG-IFN-a2b (B) conjugate in the range m / z 1200-1400.
  • Fig. 6 Thermal stability of the PEG-IFN-cc2b conjugate and unmodified IFN-a2b.
  • Fig. 7 Proteolytic stability of the PEG-IFN-a2b conjugate and unmodified IFN-a2b.
  • Fig. 8 The immunogenicity of the PEG-IFN- 2b conjugate compared with unmodified IFN-a2b.
  • Fig. 9 Pharmacokinetics of the PEG-IFN-a2b conjugate compared with unmodified IFN- a2b.
  • PEG ⁇ IFN-a2 conjugates of the formula (I), which are the object of the present invention can be used to produce medicines for preventing and / or treatment of viral diseases, because in addition to high stability and reduced immunogenicity, they have high antiviral activity.
  • pharmaceutical compositions containing as an active ingredient an effective amount of conjugate of formula (I) produced by well-known pharmaceutical methods can be used separately or in conjunction with other therapeutic agents (e.g., ribavirin) for the prevention and / or treatment of viral infections (e.g. chronic active hepatitis (particularly hepatitis B and C) (Jay et al, 2006; McHutchison et al, 2009).
  • the object of the present invention is also a way to prevent and / or treat viral diseases such as hepatitis C and hepatitis B, including introduction of a therapeutically effective amount of the claimed conjugate of formula (I).
  • IFN-a and PEGylated interferon-a can be used as immunomodulating agents for treatment of cancer, in particular, leukemic reticuloendotheliosis, laryngeal papillomatosis, 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).
  • the objects of the present invention are also medicines and pharmaceutical compositions containing the declared conjugate PEG-IFN-a in an effective quantity, possessing antiproliferative and immunomodulatory effects, which can be used to prevent and / or treat cancer and diseases associated with primary or secondary immunodeficiency, as well as the object of the present invention is a method of prevention and / or treatment of cancer and diseases associated with primary or secondary immunodeficiency, including introduction of therapeutically effective amount of conjugate PEG-IFN-a of the formula (I).
  • Conjugate of the formula (I) with an optional pharmaceutically acceptable filler, diluents and / or pharmaceutically acceptable excipients is administered intravenously, subcutaneously, intramuscularly or via any other suitable routes, for example, in the form of capsules, syrups, sprays, drops, injections or suppositories.
  • Route of administration vary depending, for example, on symptoms and age. Frequency of administration and the interval between the injections vary depending on the disease and its severity or the aim of administration (therapeutic or prophylactic use).
  • An effective amount of PEG-IFN- ⁇ is selected according to the abovementioned factors.
  • compositions with PEG-IFN-a may be included into a pharmaceutical composition with PEG-IFN-a: buffer salts (e.g. acetate, citrate, hydrocarbonate, phosphate buffers), stabilizers (e.g., polysorbate, EDTA, polyvinyl pyrollidone, dextranes, human serum albumin, ethers paraoxybenzoic acid, alcohols (e.g.
  • buffer salts e.g. acetate, citrate, hydrocarbonate, phosphate buffers
  • stabilizers e.g., polysorbate, EDTA, polyvinyl pyrollidone, dextranes, human serum albumin, ethers paraoxybenzoic acid, alcohols (e.g.
  • benzyl alcohol phenols, sorbic acid), antiseptic components (e.g., benzyl alcohol), a regulator of osmotic pressure (for example, sodium chloride, potassium chloride, polyols, for example, glycerol, arabitol, sorbitol, mannitol, lactose, dextran), surfactants (e.g., HSA, polyvinylpyrrolidone, lecithin, polysorbate 80, polyoxyethylene-polyoxypropylene copolymer, casein), surface-active substances (eg, block copolymers of ethylene oxide and propylene oxide, propylene oxide and ethylene oxide, sorbitan monolaurate, sorbitol ester, polyglycerol fatty acid ester, cocamide DEA lauryl sulfate, alkanolamide, stearyl polyoxyethylene propylene glycol, lauric ether polyoxyethylene, polyoxyethylene cetyl ether, poly
  • the diluted solution produced in example 1 was placed on a column with a cation- exchange adsorbent (CM sepharose, 300 ml), equilibrated with 5 mM sodium acetate buffer, pH 5.0, (buffer A) at the rate of 5 ml per min.
  • CM sepharose a cation- exchange adsorbent
  • the adsorbent column was washed consecutively with buffer A and buffer A containing NaCI concentration gradient from 0,05 to 0,2 M. Aliquots were sampled from each fraction for the purpose of sample analysis using the electrophoresis method in polyacrylamide gel as described in example 1.
  • the fractions that contained the monoPEGylated PEG-IFN-a2 conjugate were combined, dialyzed with 10 volumes of 20 mM sodium acetate buffer, containing 150 mM sodium chloride, then sterile filtration was performed and the resulting solution was stored at 4 ⁇ 2 °C.
  • the diluted solution produced in example 2 was placed on a column with a cation- exchange adsorbent (SP sepharose, 50 ml), equilibrated with 5 mM sodium acetate buffer, pH 5.0, (buffer A) at the rate of 5 ml per min.
  • the adsorbent column was washed consecutively with buffer A and buffer A containing NaCI concentration gradient from 0,03 to 0,3 M. Aliquots were sampled from each fraction for the purpose of sample analysis using the electrophoresis method in polyacrylamide gel as described in example 1.
  • the fractions that contained the monoPEGylated PEG-IFN-a2 conjugate were combined, dialyzed with 10 volumes of 20 mM sodium acetate buffer, containing 150 mM sodium chloride, then sterile filtration was performed and the resulting solution was stored at 4 ⁇ 2 °C.
  • the PEG-IFN-a2 conjugate produced in example 3 was diluted with 20 mM sodium acetate buffer, pH 5.0, down to the concentration of 0,1 mg per ml and 100 ⁇ of this sample were placed on the Symmetry CI 8 column (4.6x150 mm). The assay was performed at 214 nm using the "Breeze" chromatograph manufactured by the Waters Company. Based on results represented on Fig. 2 we may conclude that, according to RP-HPLC findings of, the purity of the claimed PEG-IFN-a2 conjugate is over 99%.
  • the concentration of bacterial endotoxins (BE) in samples of the PEG-IFN-cc2 conjugate produced in accordance with examples 3 and 4 was determined in vitro by means of a LAL test (gel-clot method version) in accordance with requirements of European Pharmacopoeia 6.0 Article 2.6.14.
  • a diagnostic kit manufactured by Associates of CAPE COD, Inc., LAL reagent with sensitivity of 0,03 EU per ml, Endotoxin Reference Standard (0,5 ⁇ g in a vial) and water for the LAL test were used in the assay. Based on results represented in table 1 we may conclude that BE content in conjugate sample is less than 1,5 endotoxin units (EU) per mg of protein, which is much lower than the BE level that is allowed for drugs based on recombinant proteins.
  • EU endotoxin units
  • a sample of the PEG-IFN conjugate produced in accordance with example 3 was analyzed using the electrophoresis method in polyacrylamide gel as described in example 1.
  • the electrophoresis was performed with load of 40 ⁇ g of protein per well under non-reducing conditions. Protein coloring in the gel was performed with Coomassie R-250 dye. Simultaneously, electrophoresis of unmodified IFN-a2 was performed.
  • the PEG-IFN conjugate was represented with one band (Fig. 3, track 1). Based on electrophoregrams of the PEG-IFN-a2 conjugate and of unmodified IFN-cc2, as represented on Fig.
  • the mass spectra were obtained on Ultraflex II BRUKER (Germany) MALDI-TOF mass spectrometer equipped with a UV laser (Nd). The mass spectra were obtained in the linear positive ion mode; the average weight measurement error does not exceed 10-15 Dalton.
  • the localization of the site of PEG bond with the IFN molecule was determined by comparison of mass spectra of the PEG-IFN-cc2 conjugate and unmodified IFN-oc2 after tryptic digestion.
  • PEG-IFN-a2 conjugate tryptic digest should lack the peptide corresponding to the part of the protein, in which modification occurred. In table 2 you may see mass spectra of experimental tryptic digests of the unmodified IFN-oc and the PEG-IFN-oc2 conjugate.
  • the samples produced in accordance with example 3 and example 4 were tested for specific antiviral activity in accordance with the process described in the European Pharmacopoeia using a culture of passaged MBD cells sensitive to alpha-type interferon and a culture of vesicular stomatitis virus (VSV).
  • Table 3 presents the results of study of the antiviral activity. International reference standard was used as the reference level. Proceeding from table 3 we may conclude that, despite of the conjugation with the PEG molecule with its molecular weight of 20 000 Dalton, the antiviral activity of the produced conjugates comprises 42-45% of the activity of unmodified IFN-a2. It is necessary to note that the PEG-IFN conjugates described in the prototype method, containing PEG with molecular weight of 12 000 Dalton, possessed lower activity (28-30%).
  • PEG-IFN conjugates produced in accordance with example 3 and example 4 were diluted down to the concentration of 5 mln. IU per ml and injected intramuscularly in the dose of 200 ⁇ (1 mln IU per a mouse) into ICR mice with body weight of 20-22 g once a week for 5 weeks (5 groups with 5 mice in each group). Simultaneously, samples of unmodified IFN were prepared similarly and injected into mice at the doze of 1 mln. IU. At the end of the fifth week blood tests were taken from the mice by means of retroorbital paracentesis. Blood serum was received using the standard method.
  • Antibody titer in sera obtained after of immunization of mice with unmodified IFN and PEG-IFN conjugates was determined by means of direct ELISA technique. For this purpose 100 ⁇ of the unmodified IFN-oc2 solution with concentration of 200 ng per 100 ⁇ were placed into wells of microplates. The antisera obtained from different groups of animals were placed into the wells of the plate in series from consecutive dilutions in duplicates. For detection of the resulting immune complex, antimouse antibodies conjugated with peroxidase were used. Antibody titer after administration of unmodified IFN was 1/1024. Antibody titer after administration of PEG-IFN conjugates was 1/128. Results of the study are presented on Fig. 8.
  • the area under the curve (AUC) for the claimed PEG-IFN conjugate exceeded the respective values for unmodified IFN by more than 50 times.
  • the value of the AUC exceeded the IFN AUC by 3-10 times only.
  • a medical agent containing an effective amount of the claimed PEG-IFN-cc2b (Example 17) is to be dispensed in sterile conditions into syringes from neutral glass of the 1 st hydrolytic class with brazed-in needles covered with elastic or hard protective caps, sealed with nozzles on butyl rubber plungers laminated with a fluoropolymer with the volume of 0,5, 0,8, 1 ,0 and 1 ,2 ml (for 100 ⁇ g per ml dosage), 0,5, 0,6, 0,75 and 0,9 ml (for 200 ⁇ g per ml dosage), 0,5, 0,6, 0,8, 1 ,0 and 1,2 ml (for 300 ⁇ g per ml dosage).
  • Example 19
  • a medical agent containing an effective amount of PEG- interferon alpha-2b (Example 17) is to be dispensed in sterile conditions into vials from neutral glass of the 1 st hydrolytic class sealed with fluoric rubber or nozzles on butyl rubber covers with teflon coating tightened with aluminium caps with the volume of 0,5, 0,8, 1,0 and 1,2 ml (for 100 ⁇ g per ml dosage), 0,5, 0,6, 0,75 and 0,9 ml (for 200 ⁇ g per ml dosage), 0,5, 0,6, 0,8, 1,0 and 1,2 ml (for 300 ⁇ g per ml dosage).
  • kits including a containerized medicinal agent containing PEG-IFN-a2b
  • the kit contains 1 or 4 syringes together with plungers (1 or 4 accordingly) / or vials an blister made of polymeric film together with a prescribing information, placed into cardboard pack.
  • Lam N.P. Pitrak D., Speralakis R., Lau A.H, Wiley T.E., Layden T.J. (1997), "Effect of obesity on pharmacokinetics and biologic effect of interferon-alpha in hepatitis C", Dig. Dis. Sci.;

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PCT/RU2010/000529 2010-07-20 2010-09-24 The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer WO2012011836A1 (en)

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KR1020137001858A KR101586372B1 (ko) 2010-07-20 2010-09-24 하나의 위치 이성질체로 표시되는 인터페론 알파와 폴리에틸렌글리콜의 안정한 신규 접합체
SG2013003801A SG187117A1 (en) 2010-07-20 2010-09-24 The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer
CUP2013000013A CU24193B1 (es) 2010-07-20 2010-09-24 El polietileno glicol conjugado de interferon alfa, representado por un isómero posicional

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CN114392237A (zh) * 2021-12-28 2022-04-26 上海允英生物医药科技有限公司 一种冻干病毒制剂及其制备方法
EP4139343A4 (en) * 2020-04-20 2024-06-12 National Research Council of Canada RECOMBINANT INTERFERON

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RU2572800C1 (ru) * 2014-09-22 2016-01-20 Закрытое Акционерное Общество "Биокад" Новый состав, содержащий конъюгат пэг и интерферон-альфа-2бета, обладающий сниженной болезненностью при введении
EA029498B1 (ru) * 2015-11-24 2018-04-30 Учреждение Белорусского государственного университета "Научно-исследовательский институт физико-химических проблем" (НИИ ФХП БГУ) ПРОТИВООПУХОЛЕВОЕ СРЕДСТВО НА ОСНОВЕ РЕКОМБИНАНТНОГО ИНТЕРФЕРОНА АЛЬФА-2b В ВИДЕ МИКРОЧАСТИЦ ДЛЯ ПАРЕНТЕРАЛЬНОГО ПРИМЕНЕНИЯ
RU2678332C1 (ru) 2017-09-08 2019-01-28 Общество с ограниченной ответственностью "Саентифик Фьючер Менеджмент" (ООО "СФМ") Пегилированный интерферон лямбда, обладающий высокой биодоступностью при пероральном применении, и способ его получения

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EP4139343A4 (en) * 2020-04-20 2024-06-12 National Research Council of Canada RECOMBINANT INTERFERON
CN114392237A (zh) * 2021-12-28 2022-04-26 上海允英生物医药科技有限公司 一种冻干病毒制剂及其制备方法
CN114392237B (zh) * 2021-12-28 2024-02-02 上海允英生物医药科技有限公司 一种冻干病毒制剂及其制备方法

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DOP2013000002A (es) 2013-09-15
ECSP13012398A (es) 2013-05-31
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CU24193B1 (es) 2016-09-30
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AR087227A1 (es) 2014-03-12
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