EA029942B1 - Stable pharmaceutical composition based on conjugates of biologically active proteins with polyethylene glycol containing an azo group - Google Patents

Abstract

The invention is related to the field of medicine and pharmaceutical industry and provides a pharmaceutical composition in a liquid or liophilized dosage form that contains pegylated protein in accordance with formula (I), wherein polyethylene glycol fragments are covalently connected to a protein molecule by means of azo groups:where Prot is a biologically active protein; A is an amino acid fragment of histidine; B is an amino acid fragment of tyrosine; n is an integer in an interval of 10 to 1200; m is an integer in an interval of 0 to 10; k is an integer in an interval of 0 to 10, wherein m≠k if m or k are equal to 0, in the form of a conjugate or a mixture of conjugates with different values of n, m and k, and pharmaceutically acceptable excipients selected from cosolvents, salts, acids, bases, antioxidants, preserving agents, amino acids, proteins, carbohydrates, polymers, detergents and complexons. The technical result is broadening the range of medicinal products.

Description

The invention relates to the field of medicine and the pharmaceutical industry and provides a pharmaceutical composition presented in a liquid or lyophilized finished dosage form containing a pegylated protein corresponding to formula (I), in which polyethylene glycol fragments are covalently attached to a protein molecule through azo groups:

029942 Β1

where rgo! - biologically active protein; A - amino acid fragment of histidine; In the amino acid fragment of tyrosine; η is an integer in the range from 10 to 1200; t is an integer in the range from 0 to 10; k is an integer in the range from 0 to 10, with m ^ k, if m or k is 0, in the form of a conjugate or mixture of conjugates with different values of η, m and k, as well as pharmacologically acceptable auxiliary ingredients selected from cosolvents, salts , acids, bases, antioxidants, preservatives, amino acids, proteins, carbohydrates, polymers, detergents and complexones. The technical result consists in expanding the Arsenal of drugs.

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The technical field to which the invention relates.

The invention relates to the field of medicine and the pharmaceutical industry and relates to a stable liquid or lyophilized pharmaceutical composition containing a pegylated biologically active protein in the form of a conjugate, in which a polyethylene glycol fragment is covalently attached to a protein molecule by means of an azo group. The proposed composition is used for the treatment and prevention of diseases, which shows the use of the corresponding protein, as well as an analytical standard.

Prior Art Information

Recently in medicine, conjugates of polyethylene glycol (PEG) with biologically active proteins, in which PEG is covalently attached through a suitable linker to a protein molecule, are finding increasing use. Such conjugates have several advantages compared with native proteins, since PEG protects the protein from enzymatic degradation in the body, reduces its immunogenicity, improves tolerability, and also increases the overall size of the molecule. As a consequence, it is difficult for the conjugate to be excreted through the kidneys and its circulation time in the body is increased, which results in a rarer administration of the drug and more even maintenance of therapeutic concentrations in the blood [R. Ugeieke RM, Cancers! C. "RESU1a1yuy, kissekGi1 arrgoasP ίο bgad bePuegu." Aegis Eysoe. Tobau. 2005. Νο. 10 (21). R. 1451-1458; Kuai 8.M., Mayouash S., \ Wapd X., Nabblieioi Ό.Μ., Vgaupep Ό.Τ "Lbuapsek ίη RESU1a1yi oG iproPiPyulyesytolesi1ek: ppegugu acres! K". Yehreg! Ορίη. Aegis Eiu. 2008. No. 5 (4). R. 371-383].

The biological and physico-chemical properties of such conjugates depend not only on the size and spatial structure of the attached polymer and the nature of the protein, but also on the chemical nature of the linker, which is the link between the polymer and protein fragments of the conjugate.

The finished dosage forms (GLP) of pegylated proteins should have a composition that not only excludes degradation and loss of the biological activity of the protein portion of the conjugate, but also prevents the rupture of covalent bonds between the polymer and the protein during prolonged storage. The release of free protein in the dosage form as a result of the destruction of the conjugate during storage leads to a sharp increase in the biological activity of the drug, which means the discrepancy between the claimed regulated parameters, the likely increase in reactogenicity and increased side effects. In addition, in the process of destruction of the conjugate, PEG is released in the form of various derivatives, and in some cases it is possible to accumulate various products of degradation of the linker part of the conjugate molecule, including the toxic one.

Degradation of the finished dosage forms of covalent conjugates of biologically active proteins includes not only chemical changes (oxidation, hydrolysis, etc.), but also physical changes (aggregation, denaturation, adsorption on the surfaces of the primary packaging, change in conformation, etc.). Moreover, if the rate of chemical changes increases with increasing temperature, then undesirable physical changes can occur both at elevated and at low temperatures, for example, during freezing. The problem of the stability of pharmaceutical compositions of pegylated proteins is further aggravated by the relatively small (microgram, nanomolar) content of the active substance in the dosage form. As a result, any undesirable process, for example, adsorption on the surface or oxidation by air of oxygen (including dissolved), in contrast to more concentrated dosage forms, affects the properties of the preparation especially noticeably.

On the modern pharmaceutical market there is a fairly wide range of drugs based on pegylated proteins for the treatment of a wide variety of diseases.

For example, the active substance of the drug "PegIntron" (manufacturer - с Ь Ь ег ег-Р 1)) is a pegylated interferon a-2b (PEG-INF-a-2b, INN: peginterteron alpha-2b), in which a linear PEG of 12 kDa is covalently attached to the amino group INF a-2b through the carbamate bond (WD 1995/013090, publ. 05/18/1995). The drug is available in the form of a lyophilisate containing, in addition to the active ingredient PEG-INFA a-2b, sodium hydro- and dihydrophosphates, sucrose and polysorbate 80 (№О 1999/048535, publ. 30.09.1999).

The active substance of the drug "Pegasis" (manufacturer - R. NoGGthii-Ba CosBe IB.) Is pegylated interferon a-2a (PEG-INFA a-2a, INN: peginterferon alpha-2a) in which the branched PEG with a mass of 40 kDa is covalently attached to the amino group of INF a-2a through an amide bond. The PEG moiety is a dipegylated lysine in which two linear PEG molecules of 20 kD each are covalently attached by a carbamate bond to the lysine amino groups (CI 97108698, publ. 10.05.1999). The drug is available in the form of a solution containing, in addition to the active substance PEG-INFA a-2a, sodium chloride, benzene alcohol, acetic acid, sodium acetate and polysorbate 80 (I8 5762923, publ. 09.06.1998). These drugs are used to treat viral hepatitis.

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The active substance of the drug Mircera (manufacturer - R. NoGGtapp-la Rosier L1.) Is a pegylated erythropoietin beta (PEG-EPO β, INN: methoxy polyethylene glycol-epoetin beta) in which a linear PEG-butane acid of 30 kDa is covalently added the amino group of erythropoietin through an amide bond (AO 2002/049673, publ. 27.06.2002). The drug is available in the form of a solution containing, in addition to the active ingredient PEG-EPO β, sodium sulfate, sodium dihydrophosphate, L-methionine, mannitol and poloxamer 188 (Pigos p68) (AO 2001/087329, publ. 22.11.2001). This hematopoietic stimulating drug is used to treat anemia in chronic renal failure.

The active substance of the drug "Neulam" (producers - R. NoGGtapi-la Rossier LJ, Atdep 1ps.) Is a pegylated granulocyte colony-stimulating factor (PEG-G-CSF, INN: Pagfilgrastim), in which a linear PEG with a weight of 20% is intact. the amino group of G-CSF through a secondary amine bond (JSC 1996/011953, publ. 04/25/1996). The drug is available in the form of a solution containing, in addition to the active ingredient PEG-G-CSF, sorbitol, sodium acetate and polysorbate 20 (RyukUap Raskade 1pkeg1 Nyak! A ™ (RedGydggyt), I8 20090247450, publ. 01.10.2009). This drug is used as a stimulator of leukopoiesis in neutropenia.

The active substance of the drug "Somavert" (manufacturer - RP / Eg 1ps.) Is a pegylated growth hormone receptor antagonist (INN: Pegvisomant), in which from 4 to 6 linear PEG fragments with a mass of 5 kDa are covalently attached to the lysine amino groups of the protein through an amide bond (I8 5849535, publ. 12/15/1998). The drug is available in the form of a lyophilisate containing, in addition to the active ingredient, glycine, mannitol, sodium hydro- and dihydrophosphates (ΝΏΑ 21-106 Cotau Rrróей Rayay Leye !, 25/03/2003).

This drug is used to treat patients with acromegaly, in which surgery and / or radiation therapy were ineffective.

Most modern commercial preparations based on pegylated proteins are conjugates in which the polymer fragment is attached to the amino groups of biologically active proteins through an appropriate linker. However, at present, conjugates in which the amino groups of proteins are not affected are also of interest. Preparations based on such active substances may have a different ratio and the number of positional isomers, which may have a positive effect on the biological activity and reactogenicity, and ultimately on the overall pharmacological value of the drug. Such compounds, for example, include biologically active conjugates, where the polymer is attached to the aromatic fragments of protein molecules, for example, by means of the azo group. In particular, such conjugates include pegylated proteins, in which the linker between the polymer and protein fragments in the molecule is 4-diazenyl benzoate. In their chemical nature, these conjugates are polyethylene glycol esters of 4-diazenyl benzoic acid, in which the azo group is attached to the tyrosine or histidine aromatic fragments of the corresponding protein. In these conjugates, the biologically active protein can be interferon α, β or γ (CB 2433135, publ. November 10, 2011), erythropoietin (CB 2433134, publ. November 10, 2011), human growth hormone (ЕА 201101664, publ. 30.11.2012) , granulocyte colony-stimulating factor of a person (ЕА 201101663, publ. 28.06.2013) .. Dosage forms based on the above conjugates and their composition are not described in the literature.

A known disadvantage of azo compounds is their susceptibility to reducing and oxidative degradation induced by many factors. The rate of destructive changes depends on pH, temperature, light intensity, the presence of certain excipients. Using the example of azo dyes of various technical classes, it was shown that the photochemical almost complete degradation of the azo compound can be achieved by direct exposure to UV radiation, regardless of the concentration of the substrate in the solution (S. Ootek ya 8yua, 1. kk Rapa. "Ryoyyyet1sa1yyyyyo! a / o yue sh adieoik koiii lu iyu ygayaPop ". 1. Ryoisiet. apy RyoYo1. A: SjetYgu. Vo1. 155 (2003). P. 133-143).

The impact of ultrasound, leading to the formation of hydroxyl radicals, is also capable of destroying the molecules of some azo compounds, however, the transformations do not proceed quantitatively (Taileg M.M., Sieb / O.M.A. Kyogek A. "BedgayopoG A / o Buek lu Lassake apy Ingakoipy Tgea1tep1 ". Arr1. Epen1gop. Myugoyuk Wo1. 71, Νο. 5 (2005). R. 2600-2607).

Azo compounds also undergo restorative destruction in the environment of the gastrointestinal tract. Hydrophilic Acceptance Ayuapsei Vyutayya1k w Vyteyuya1 Epdeteppda apy Bgid Beyuegu, 11 (1996. R. 349-350).

Without being limited to a particular theory, the inventors believe that the presence of an azo group conjugated with aromatic fragments in the structure of the conjugates makes it possible to add an additional 2 029942

An integral p ^ n * electron transition leads to the appearance of an absorption band in the visible region of the electronic spectrum. This is facilitated by the presence of electron-donating (for example, phenolic hydroxyl tyrosine) and electron-withdrawing (ester carbonyl) substituents in various aromatic fragments of the conjugate. As a result, a significant proportion of the molecules of the considered conjugates are capable of passing into an unstable excited energy state upon absorption of photons in the visible region of the spectrum, followed by photodestruction.

In addition, in the above pegylated proteins, the linker is diazenyl benzoate containing an easily hydrolysable ester bond. Therefore, these conjugates are not only sensitive to oxidation / reduction due to the presence of the azo group, but may also be unstable under conditions that promote hydrolysis (in particular, enzymatic). At the same time, the protein portion of the conjugate can undergo proteolysis, deamidation, disulfide exchange, sulfoxidation, oxidation, racemization, aggregation, etc., which affects the overall conformation and, therefore, the biological properties of the conjugate, such as activity, immunogenicity and reactogenicity . A noticeable course of these reactions may lead to a decrease in the pharmacological effect and an increase in undesirable side effects and toxicity.

The stability of the main active ingredient in a pharmaceutical composition strongly depends on the auxiliary components included in the composition of the medicinal product, such as buffer salts, sugars and detergents, as well as on the form of the composition itself. Derivatives of a physiologically active polypeptide, such as insulin, containing PEG fragments and paraphenylenediazonium in the molecule are disclosed in IZ 4179337 (publ. 12/18/1979), but their satisfactory stability is not confirmed by the description.

A significant disadvantage of lyophilisate is the complexity of the process of their production. The auxiliary components used for this purpose must protect the conjugate as a preparation process, i.e. during the dissolution, freezing and drying, and during storage of the SFF. In addition, lyophilization conditions — temperature, pressure, duration of freezing and sublimation — affect the stability of the active substance in freeze-dried SFFs. These factors also affect the physical state of the resulting product - amorphous glassy, amorphous soft, crystalline, or a combination of them, which determines the pharmacological properties. Another disadvantage of lyophilisates is the need to restore the dosage form before the injection, which requires additional manipulations from the medical staff and is associated with the risk of improper handling of the pharmaceutical product. In addition, the most universal composition of SFF is one that stabilizes the conjugate, which is both in solution and in the lyophilisate. This is due to the fact that if the drug is supposed to be released only as a solution, then it is desirable to produce its standard samples or analytical standards in a more stable lyophilized form, and the composition of the latter, after reconstitution with a solvent, should be identical to the composition of the analyzed preparation.

Thus, based on the highly valuable pharmacological properties of conjugates containing the azo group, the creation on their basis of a single stable pharmaceutical composition suitable for both liquid and lyophilized forms is an urgent task.

Summary of Invention

The task according to the invention is solved by creating a pharmaceutical composition presented in a liquid or lyophilized finished dosage form (SFF) containing a pegylated protein, in which polyethylene glycol fragments are covalently attached to a protein molecule by azo groups, corresponding to formula (I)

where rgo! - biologically active protein;

A - amino acid fragment of histidine;

B - amino acid fragment of tyrosine; η is an integer in the range from 10 to 1200; t is an integer in the range from 0 to 10;

k is an integer in the range from 0 to 10, with t Ψ k, if m or k is 0, in the form of a conjugate or a mixture of conjugates with different values of η, m and k,

as well as pharmacologically acceptable auxiliary ingredients selected from cosolvents, salts, acids, bases, antioxidants, preservatives, amino acids, proteins, carbohydrates, polymers, detergents and complexones.

Preferably, the biologically active protein is selected from recombinant human proteins, such as, for example, interferon and erythropoietin.

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More preferably, the biologically active protein is erythropoietin and the composition is presented as a liquid SFF.

It is also more preferable if the biologically active protein is an interferon and the composition is presented in the form of a lyophilized SFF.

Alternatively, it is more preferable if it contains from 0.01 to 50 mg of a conjugate of formula (I), contains 5 to 100 mg of mannitol as a carbohydrate, contains 0.1 to 20 mg of glycine as an amino acid, and contains 0 as an antioxidant , 1 to 10 mg of b-methionine, contains 0.01 to 1 mg of polysorbate 80 as detergent, contains 0.01 to 1 mg of ethylenediaminetetraacetic acid as complexone, and contains 1 to 25 mg of sodium dihydrogen phosphate as a salt anhydrous salt, sodium hydroxide as base or sodium hydroxide as base You introduced acetic acid or hydrochloric acid to a pH value of 3.5 to 7.0 and as a solvent contains water for injection up to 1 ml.

The technical result of the invention is to expand the Arsenal of pharmaceutical compositions containing as chemically active proteins that are chemically modified to increase the time of their circulation in the bloodstream.

The proposed pharmacological composition can be presented in the form of a solution or in the form of a lyophilisate, which can be converted into a liquid form by adding an appropriate pharmacologically acceptable solvent and, optionally, a cosolvent. The composition may contain auxiliary substances, such as pharmacologically acceptable salts, introduced into it as such or formed during its preparation, acids or bases, preservatives, amino acids, proteins, carbohydrates, detergents, polymers, chelating agents.

Pegylated Pro Rgo! may be a protein or polypeptide, such as interferon α, interferon β, interferon γ, interferon δ, erythropoietin α, erythropoietin β, human growth hormone, growth hormone receptor antagonist, granulocyte colony-stimulating factor, tumor necrosis factor, interleukin-1 (α or β ), interleukin-2,3,4,5,6,7,8,9,10,11,18,33, uricase, L-asparaginase.

Pharmacologically acceptable solvent can be water for injections, bacteriostatic water for injections, isotonic solutions of glucose or sodium chloride, Ringer, Ringer-Locke and Ringer-Tyrode solutions in various physiologically acceptable variants, Disol, Trisol, Acesol, "Chlosol", "Kvartasol", "Lactasol", Petrov's blood-replacement fluid.

A pharmacologically acceptable co-solvent can be ethanol, propylene glycol, glycerin, glycofurol, fatty oils, such as peach, almond, soybean, and Kosher HB ™ (manufactured by ΒΑδΡ Comp., ΕΛδ 61791-12-6), which is polyoxyethylated castor oil.

Pharmacologically acceptable salts can be phosphates, acetates, citrates, succinates, adipates, lactates, fumarates, malates, tartrates, benzoates, 4-aminobenzoates, carbonates, bicarbonates, chlorides, bromides, and sodium, ammonium, calcium, magnesium, aluminum sulfates, 2 -amino-2-hydroxymethylpropane-1,3-diol (trometamol) or potassium of various degrees of substitution, if they can exist, are physiologically tolerable and have acceptable solubility, as well as their mixtures, hydrates and optical isomers, if their formation is possible.

Pharmacologically acceptable acids can be phosphoric, acetic, citric, succinic, adipic, lactic, fumaric, malic, tartaric, benzoic, 4-aminobenzoic, hydrochloric and sulfuric, as well as their mixtures, hydrates and optical isomers, if their formation is possible.

Pharmacologically acceptable bases are sodium, ammonium, potassium, magnesium, calcium, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (trometamol) hydroxides or mixtures thereof.

Pharmacologically acceptable antioxidants are ascorbic acid, cysteine, acetylcysteine, glutathione, unithiol, dithiothreitol, methionine, tocopherols, tocotrienols, retinol, their salts, esters, and optical isomers, if their existence is possible, as well as sulfur-containing, 4-ray and 4-yarn salts, ethers, and optical isomers, as well as for the sulfur, 4-yarn, and 4-yarn, and for example, for the use of sulfur, 4-yarn, 4-yarn, 4-yarn and 4-optics isomers and optical isomers, if necessary, as well as pyroacetate, and 4-yarn, as well as 4-yarn, 4-yarn, 4-yarn, 4-yarn, 4-yarn salts, esters and optical isomers, if they are possible, as well as sulfur-containing compounds, 4-yarn, 4-yarn, 4-yarn and 4-yarn are used. In addition, to prevent oxidation, the dosage form may be in an atmosphere of inert gases such as argon, nitrogen, or a mixture thereof.

Pharmacologically acceptable preservatives are phenol, cresol, chlorocresol, benzyl alcohol, 4-hydroxybenzoic acid esters, sodium ortho-ethyl mercine, sodium (merthiolate), chlorobutanol hydrate.

Pharmacologically acceptable amino acids include glycine, alanine, p-alanine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, glutamine, glutamic acid, asparagine, aspartic acid, threonine, serine, homoserine, cysteine, cystine, phenylalanine, histidine, lysine, proline, hydroxyproline, arginine, methionine, carnitine, ornithine, sarcosine, taurine or their salts or their mixture.

Pharmacologically acceptable proteins are albumin, gelatin, gelatosis and various physiologically acceptable protein hydrolysates, including those suitable for parenteral nutrition, and their mixture.

Pharmacologically acceptable carbohydrates are glucose, fructose, mannitol, sorbitol, sucrose, trehalose, tetragalose, meglumin, dextrans, cyclodextrins, hydroxyethyl starch, sialic acids, or a mixture thereof.

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Pharmacologically acceptable detergents are polysorbates, poloxamers, proxanol, phospholipids, lecithin, or a mixture thereof.

Pharmacologically acceptable polymers are polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, or a mixture thereof.

Pharmacologically acceptable chelating agents are ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, triethylenetetraamine hexaacetic acid or their salts or a mixture thereof.

More preferably, the present invention provides a pharmaceutical composition, which includes:

from 0.01 to 50 mg of a conjugate of formula (I); 5 to 100 mg of mannitol; from 0.1 to 20 mg of glycine; from 0.1 to 10 mg of L-methionine; from 0.01 to 1 mg of polysorbate 80;

from 0.01 to 1 mg of ethylenediaminetetraacetic acid; from 1 to 25 mg of sodium dihydrogen phosphate in terms of anhydrous salt; sodium hydroxide or acetic acid or hydrochloric acid to pH from 3.5 to 7.0; water for injection - up to 1 ml.

This pharmaceutical composition is prepared in the form of a solution by dissolving all components in water for injection and subsequent sterilizing filtration. If necessary, a sterile solution can be poured under aseptic conditions in syringes, vials, cartridges or ampoules, as well as frozen and dried.

The pharmaceutical composition prepared in accordance with the invention can be administered to humans and animals or used for analysis, for example, as an analytical standard. The composition is used in the form of a standard dosage form intended for parenteral, intranasal, instillation, insufflational and application administration. This composition can be presented in the form of a solution, powder and lyophilisate for injection, as well as in the form of a powder, lyophilisate, solution and spray for local and external use. The lyophilisate, powder and injection solution of the present invention can be administered using conventional injection devices, such as syringes, allowing the administration of from 0.1 to 50 ml, as well as an infusion up to 1000 ml of the composition as a standard dose. Lyophilisate and solution for local and external use can be administered in the form of drops, irrigation or lubrication, for example, to treat the mucous surfaces of the throat, eyes, nose, and ear.

The pharmaceutical composition according to this invention can be presented in the form of a lyophilisate for preparing a solution for intramuscular and external use, a lyophilisate for preparing a solution for intracavitary and external use, a lyophilisate for preparing a solution for injection; a lyophilisate for preparing a solution for intravenous administration, a lyophilisate for preparing a solution for intracutaneous administration, a lyophilisate for preparing a solution for intramuscular administration, a lyophilisate for preparing a solution for intramuscular and subcutaneous administration, a lyophilisate for preparing a solution for infusions, a lyophilisate for preparing a solution for injection in pediatrics, a lyophilisate for preparing a solution for injection and intravesical administration, a lyophilisate for preparing a solution for injection and inhalations, lyophilisate for solution for injection and topical use, lyophilisate for solution for injection and cutaneous scarification application, lyophilisate for solution for injection and topical use, lyophilisate for solution for local and external use, lyophilisate for the preparation of eye drops, a lyophilisate for the preparation of a nasal solution, a lyophilisate for the preparation of a nasal solution in pediatrics, a lyophilisate for the preparation of a solution d For inhalation, lyophilisate for preparing a solution for external use.

The pharmaceutical composition according to this invention can also be presented in the form of an aerosol for inhalation, metered, aerosol for topical use, aerosol of nasal, aerosol of nasal metered, aerosol for external use.

Alternatively, the pharmaceutical composition according to this invention can be presented in the form of drops for local use, eye drops, nasal drops, eye drops and nasal drops, eye drops and ear drops, mouth rinse drops, nasal drops in pediatrics, nasal drops and ear drops, ear.

Alternatively, the pharmaceutical composition according to this invention can also be presented in the form of a powder for inhalation, a metered dose, a powder for preparing a solution, a powder for preparing a solution for intraocular administration and eye drops, a powder for preparing a solution for injection, a powder for preparing a solution for intravenous administration, powder for preparation of solution for intravenous and intramuscular administration, powder for preparation of solution for intramuscular administration, powder for preparing Lenia solution for intramuscular and

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subcutaneous injection, powder for preparing the solution for intracavitary injection, powder for preparing the solution for infusions, powder for preparing the solution for subcutaneous administration, powder for preparing the solution for injection and topical use, powder for preparing the solution for local and external use, powder for preparing vaginal solution, powder for preparing eye drops, powder for preparing solution for endotracheal administration, powder for preparing solution for n ruzhnogo application, a powder for the preparation of drops in pediatrics.

Further, the pharmaceutical composition according to this invention can be presented in the form of a concentrate for preparing a solution for injection, a concentrate for preparing a solution for intravenous administration, a concentrate for preparing a solution for external use, a solution for intraperitoneal administration, a solution for intravenous administration and inhalation, a solution for intradermal administration and topical use, solution for inhalation, solution for injection, solution for intravenous administration, solution for intravenous and extra trimuscular injection, solution for intraocular administration, solution for intracutaneous administration, solution for subcutaneous administration, solution for intracutaneous and subcutaneous administration, solution for intramuscular administration, solution for intramuscular and subcutaneous administration, solution for intravascular administration, solution for infusions, in particular - dextrose solution, in a solution of sodium chloride 0.9%, solution for infusions frozen, solution for injections in pediatrics, solution for injections and external use, solution for the skin scarification application, solution for perfusion, solution for subconjunctival administration and instillation into the eye, solution for endolymphatic administration, solution for topical use, solution for external use, nasal solution, rectal solution, solution for external use of foaming, solution for external use and preparation of medicinal forms, solution for peritoneal dialysis, liquid for inhalation.

Further, the pharmaceutical composition according to this invention can also be presented in the form of a spray for local use, a spray for topical administration, a dosed spray, a nasal spray, a nasal spray in pediatrics, a nasal spray dosed, a nasal spray dosed in pediatrics, a nasal forte spray, an outdoor spray, spray powder.

The proposed composition in the form of solutions are stable in accordance with the requirements of current regulatory documents for 3 years and 6 months when stored at a temperature of 2 to 8 ° C in a dark place, which is confirmed by the research results given in table. 1-4.

The single and repeated liquid compositions in accordance with the invention in human doses and fivefold human doses do not show acute and chronic toxicity, which is characterized by the absence of signs of inflammatory, dystrophic and necrotic processes in the internal organs of animals according to the results of histological studies and planned necropsy, as well as not Significant differences are found in the indices of general and biochemical blood analysis between the experimental and control groups of animals.

Compositions in the form of solutions in accordance with the invention do not have a local irritating effect on the eyes and do not cause allergic hypersensitivity reactions of the immediate and delayed types.

The results of preclinical studies show that the developed liquid pharmacological composition is non-toxic, pyrogen-free, does not adversely affect the systems and organs of laboratory animals.

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Stability of pegylated erythropoietin β liquid forms at 37 ° С

Table 1

£ PEG-EPO, µg / ml Mannitol, mg / ml one one Glycine, mg / ml Methionine, mg / ml Polysorbate 80 mg / ml EDTA, mg / ml ί Content (%) by 1 week 2 weeks 3 weeks 4 weeks 5 weeks | Aggregates | | Oligoforms | PEG-EPO | epo | Aggregates | | Oligoforms | PEG-EPO | epo | Aggregates | Oligoforms | PEG-EPO | epo | | Aggregates | | Oligoforms | PEG-EPO | EPO | | Aggregates | | Oligoforms | PEG-EPO | EPO | one 312 40 3.12 five 1.50 0.1 0.0750 7.0 04 * sch, m ⁷ SP 00 Yu SP about <L sch, sc * 84,8 about Well 00 sch sch tt ^79.6 τζ SP SP with·* <L MF t — h <L G- "L 40 00 sch 78.7 t sch 2 312 40 5.46 five 1.50 0.1 0.0750 6.0 about, r-4 00 oo eight about ’F Well about\ SP 86.4 'D 1-4 04 cn 'T sch 82.5 sch sch SP MF sch 40 cn oo about. SP 40 sch 82.9 s 3 312 40 5.46 5.0 1.50 0.10 0.0750 5.0 h oo * sch SP 00 40 00 'H SP 04 90.2 gu tg SP SP SP sc ' 89.2 sch νί <n SP SP sch | 87.2 about about 40 SP sc ' gt 00 sch, G "' four 156 20 9.36 12.5 0.75 0.05 0.0375 6.0 SP Che s about Ώ. <l * sch SP ^87.4 ABOUT\ SP about "L sch 88.4 "L tg * about\ SP 00 G "* 00 SP r ~ - * SP ’F 04 sc ' sch 40 00 40 40 five 156 20 9.36 2.5 10.75 0.05 0.0375 6.0 <34 SP sch 92.7 sch sc * about, about sch | 9‘06 1 -¾ cn 00 SP 4G sc ' | 89.2 40 SP Gu Gu 00 00 sch 40 tt " about sch SP $ 5 40 40 6 156 20 9.36 2.5 0.75 0.15 0.0375 6.0 OO 40 about sch 89.9 | SP 00 sc * 00 MF * 92.0 sch tg P; c00 04 SP <> SP tt Gu 00 oo sch, 40 tt P" su sc ' 87.0 04, "L 7 156 20 9.36 2.5 0.75 0.45 0.0375 6.0 04 H- ' - 1-4 ABOUT\ 04 G ' sc * sch 1 ⁹¹ · ⁵ ί 40 ’F G " V 3 · 00 "L, 'ί * 00 SP sch 00 oo 00 SP "L * 04 - “g * 04 "L 58 su 40 *

Stability of pegylated erythropoietin β liquid forms at 20 ° С

table 2

3 PEG-EPO, µg / ml Mannitol, mg / ml | ί 2 Glycine, mg / ml L-Methionine, mg / ml Polysorbate 80 mg / ml EDTA, mg / ml I but. Content (%) by 1 month 2 months 3 months 6 months 9 months Aggregates | Oligoforms PEG-EPO EPO I one { Oligoforms | PEG-EPO I Λ Aggregates | Oligoforms | PEG-EPO | epo | Aggregates | Oligoforms | PEG-EPO EPO Aggregates | Oligoforms | PEG-EPO EPO one 312 40 5.46 5.0 1.50 0.10 0.075 6.0 about sc * <l sch 95.0 "L about* 00 SP sch 89.2 about oo sch SP tg ^91.9 1 about. SP SP SP 92.9 | r ~ about 00 <h 04 sc ' 04 OO 00 "L 2 312 40 5.46 5.0 1.50 0.10 0.075 5.0 <h SP ^91.3 • H; about SP sc ' ^92.4 • d sch "L sc ' SP sc ' ^94.3 04 ABOUT "Y mr r ~ sch 88.6 about "L 40 sch 86.9 "L 3 156 20 9.36 12.5 0.75 0.05 0.0375 6.0 *P about 00 SP Gu "L 04 about cn h · ' about. sch 90.9 00 sc * su sch sch ^92.6 1 "P about* SP νί SP 88.3 1 <h SP 00 SP about SP 85.7 "L g- * four 156 20 9.36 2.5 0.75 0.15 0.0375 6.0 about r ~ sc * 1 ^95.1 sch 04 MF sch | 92.6 MF su tg 40, SP I 90.4 I 2 > l "L about SP I 90.2 1 cn sch, chg 00 sch I 87.0 about 40

Stability of the liquid form of pegylated erythropoietin β at 4 ° C

Table 3

Composition Month Content (%) Activity, U / ml Aggregates Olgoform PEG-EPO EPO PEG-EPO -312 mcg / ml Mannitol - 40 mg / ml Ν3Η ₂ ΡΟ4 · 2Η ₂ - 7.8 mg / ml Glycine - 5 mg / ml L-Methionine -1.5 mg / ml Polysorbate 80 - 0.1 mg / ml EDTA - 0.075 mg / ml pH6.0 3 0.8 2.2 96.2 0.8 EVIL ² 6 1.7 2.4 94.8 1.1 3-10 ² 9 5.5 3.2 91.6 0.8 3-10 ² 12 2.3 2.9 91.7 3.1 4-10 ²

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Table 4

Analytical data confirming the stability of the liquid pharmaceutical composition of PEG-EPO at the studied shelf life

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Information confirming the possibility of carrying out the invention

Further, the invention will be illustrated by the following examples, confirming the possibility of its implementation with the achievement specified in the description of the technical result.

Example 1. Preparation of PEG-EPO liquid pharmacological compositions.

In volumetric flasks with a capacity of 100 ml pre-prepared solutions of each auxiliary component, for which:

1) in a small amount of water dissolve a portion of 20.00 g of mannitol and bring the final volume to the mark with water;

2) dissolve a portion of 2.50 g of glycine in a small amount of water and bring the final volume to the mark with water;

3) in 50 ml of water dissolve the weight of 0.75 g of L-methionine and bring the final volume to the mark with water;

4) in a small amount of water dissolve a weighed 0.05 g of polysorbate 80 and bring the final volume to the mark with water;

5) dissolve a weight of 0.0375 g of EDTA in 80 ml of water and bring the final volume to the mark with water;

6) in 50 ml of water dissolve a weighed 3.90 g of ΝαΗ ₂ ΡΟ ₄ · 2Η ₂ Ο and bring the final volume to the mark with water.

Next, all solutions are filtered through a sterilizing filter with a pore size of 0.22 μm in sterile containers and stored at 2-8 ° C.

Different versions of the PEG-EPO liquid pharmacological composition are prepared by mixing the appropriate volumes of the auxiliary components solutions, adding the appropriate amount of the PEG-EPO solution with a concentration of 0.624 mg / ml and 10% ΝαΟΗ to the required pH value. Next, the solutions are brought to the appropriate volume with water, filtered through a sterilizing filter with a pore size of 0.22 μm in sterile hermetically sealed vials.

Example 2. Determination of the stability of liquid pharmacological compositions of PEG-EPO.

The bottles are divided into 3 groups and thermostatic at 4, 20 and 37 ° C. During storage, samples taken from each group are analyzed, determining the specific activity, aggregate content, oligoform, pegylated and free EPO. Sampling stored at 4 ° C is carried out 1 time per quarter, stored at 20 ° C, 1 time per month, and samples stored at 37 ° C, 1 time per week. The composition of each sample is analyzed by size exclusion HPLC on a Siregbech ™ 200 10/300 CP column (10x300 mm, 13 μm) at room temperature and the flow rate of the mobile phase (0.15 M α in 50 mM phosphate buffer, pH 7) of 0.5 ml / min Detection is carried out spectrophotometrically at 280 nm. At the same time determine the specific activity. The results are shown in Table. 5. The initial parameters are taken as 100%.

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Table 5

The stability of the liquid pharmacological composition of the PEG-EPO

Time months Definable parameter The value of the studied parameter in% of the original value 4 ° C 20 ° С 37 ° C 6 The content of the PEG-EPO 100 99 97 Aggregate content 0 one 3 Specific activity 100 98 94 12 The content of the PEG-EPO 97 97 - Aggregate content 3 3 - Specific activity 100 96 - 18 The content of the PEG-EPO 97 97 - Aggregate content 3 3 · Specific activity 100 90 -

The data confirms that the proposed pharmaceutical composition for the preparation of liquid drug "Peg-erythropoietin solution for intravenous and subcutaneous administration" is stable for 18 months.

Example 3. Determination of the specific activity of PEGEPO liquid pharmacological compositions by measuring the stimulation of the formation of reticulocytes in normocythemic mice.

2 days before the start of the study, groups are formed for each dilution, seating the mice on the P1 line (SVLxS57VE, 7-9 weeks) of 6 animals per cage. For the control on phosphate-albumin buffer solution, dilutions of samples and an international standard sample of erythropoietin (mSOE) (ΕΏΡΜ, cat. No. 1515000) with a content of 90, 30 and 10 IU / ml in a volume of 10 ml are prepared. In each group, 0.5 ml of one of the dilutions is injected subcutaneously into mice. The control mice were injected with 0.5 ml of phosphate-albumin buffer solution,

Four days after the administration, blood samples are taken from the animals (not less than 0.2 ml) and the number of reticulocytes is determined, for which 1 μl of whole blood is added to 1 ml of orange thiazole solution at a concentration convenient for determining reticulocytes. After staining for 10 min, reticulocyte counting is performed microfluorimetrically in a flow cytometer using an analysis of a red fluorescent histogram.

The work uses the international standard of erythropoietin (ECOE) (Eigorep 1) parasiticle South Pte C) and the 1st of the United States. Νο. E 151500).

For the introduction of a dilution of the studied drugs and a standard sample of erythropoietin in doses of 10.25 and 62.5 IU. The investigated samples are administered once with 0.5 ml intraperitoneally in doses of 10.25 and 62.5 U. Doses before administration are stored at room temperature for no more than 4 hours.

For research, use is made of an international standard sample of erythropoietin (EPO), a solution of the substance of erythropoietin (with a symbol - series No. 1) and a solution of pegylated erythropoietin (with a symbol - series No. 3). The estimated activity of the samples was approximately 2000 nominal IU / ml. In tab. 6 shows the data on the nominal and calculated actual doses of the studied samples of erythropoietin, as well as the corresponding correction factors.

Table 6

Nominal (in U) and real (in IU) doses and correction factors for the studied samples of erythropoietin

Sample Nominal doses, IU Estimated actual doses, IU Corrections coefficients The solution of erythropoietin (series number 1) 10.0 9.1 0.910 25.0 24.9 1,000 62.5 68.5 1,009 Medium: 1.00 + 0.05 Solution pegylated erythropoietin (series №3) 10.0 9.8 0.98 25.0 24.7 0.99 62.5 62.5 1.00 Medium: 0.99 + 0.06

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The average correction factor is close to 1.00, so the samples of Erythropoietin Series No. 1 and No. 3 in the presented estimated activity of 2000 IU / ml in their erythropoietic activity correspond to the international standard sample.

The obtained data and the results of earlier experiments on the study of the physicochemical properties of pegylated erythropoietin indicate that the size of polyethylene glycol attached to erythropoietin slightly shields the active sites of EPO in the synthesized conjugates and therefore the specific activity in the preparation is maintained at rather high levels. This is further confirmed by a comparison of the proposed drug PEGeritropoetin with a commercial drug pegylated EPO Mircera and an international standard sample of erythropoietin. The results of the experiments are presented in table. 7

Table 7

The results of the comparison of the specific activity of various drugs made on the basis of pegylated erythropoietin

A drug Specific sample activity, IU / mg protein % of the specific activity of the sample, IU / mg protein International EPO Standard * 141Ί0 ³ 100 Peg-erythropoietin _{34 10} l 24 Mircera _{2θ 1θ3} 14

* Hegueroep BEP E1515000, 35280 reg U1a1.

As can be seen from the table. 7, the residual specific activity of the liquid drug formulation of the PEG-erythropoietin compound according to the invention in terms of the activity of native erythropoietin is about 30-35%, which exceeds the specific specific activity of the commercially produced Myrcera pegylated erythropoietin. Experimental production series of the drug "PEG-erythropoietin" for 3 years and 6 months. retain the original specific activity when stored at a temperature of from 2 to 8 ° C in a place protected from light.

Example 4. Preclinical study of the toxic effects of the PEG-EPO liquid pharmacological composition.

A. Determination of the acute toxicity of a PEG-EPO liquid pharmaceutical composition.

The study of acute toxicity is carried out on rabbits (25 animals) and mice (50 animals), divided into 5 experimental groups of animals of each animal species (5 males and 5 females in each group). Animals of the 1st and 6th groups are injected with a solvent (water for injection). Animals of the 2nd, 4th, 7th and 9th groups are injected with PEG-EPO liquid pharmacological composition in doses of 1.5 and 7.5 mg / kg, respectively: in the 2nd and 4th groups - intravenously, animals 7 and 9- th groups - subcutaneously. Animals of the 3rd, 5th, 8th and 10th groups receive the comparison drug Mircera in doses of 1.5 and 7.5 µg / kg, respectively: in the 3rd and 5th groups - intravenously, animals of the 8th and 10th groups - subcutaneously. Thus, in studies, therapeutic and fivefold therapeutic doses for humans are administered.

On the 15th day of the study, animals are subjected to euthanasia, followed by examination of the macrodamages of organs, and sampling is carried out for hematological, biochemical, and histological studies. As a result of the studies, it was proved that the use of the PEG-EPO liquid pharmacological composition and the Mircera comparison drug in the study of acute toxicity does not cause animal death, does not lead to weight loss, the formation of inflammatory changes and the development of necrobiotic processes at the injection site. This indicates the absence of symptoms of intoxication.

B. Determination of the chronic toxicity of the liquid pharmacological composition of PEG-EPO.

Chronic toxicity studies are performed on mice (40 animals), guinea pigs (80 animals) and rabbits (20 animals) from 16 experimental groups. Animals in groups with odd numbers are injected with a PEG-EPO liquid pharmacological composition, animals in groups with even numbers are given the comparison drug Mircera. Animals from the 1st to the 8th group receive the study drugs at a dose of 2.5 mg / kg, animals from the 9th to the 16th group receive a dose of 5 mg / kg. Animals in 1-, 2-, 3-, 4-, 9-, 10-, 11-, 12th groups, the drugs are administered intravenously, in 5-, 6-, 7-, 8-, 13-, 14-, 15th, 16th groups - subcutaneously for 6 weeks (42 days) twice a week. Thus, in studies, therapeutic and two-fold therapeutic doses for humans are administered.

On the 29th day of the study and on the 14th day after the end of the injection of the PEG-EPO liquid pharmacological composition and the comparator drug (the 56th day of the study), the animals were euthanized and examined for macroscopic organs, they were sampled for hematological, biochemical and histological studies. The use of the studied drugs does not cause the death of animals, reduce motor and nutritional activity, changes in physiological functions, weight loss and internal organs, which indicates the absence of a toxic effect of the drug on the systems and organs of laboratory animals.

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In planned necropsy, pathological and histological studies performed show that a single or repeated administration of PEG-EPO and Mircera in the doses studied does not cause the development of pathological, including inflammatory, dystrophic and necrotic processes in the internal organs of animals.

The results of the analysis of hematological and biochemical parameters of the blood of laboratory animals in the "acute" and "chronic" experience show the absence of pronounced pathological changes in the indicators of general and biochemical blood analysis, as well as the absence of significant differences between the experimental and control groups of animals.

The drug "PEG-EPO" is pyrogen-free, as it is administered intravenously to rabbits at a dose of 5 µg / 2.5 kg, since the sum of the temperature increases does not exceed 1.2 ° C.

Example 5. Determination of local and local irritating action of the liquid pharmacological composition PEG-EPO with subcutaneous and intravenous administration.

A. Local action.

Local action after subcutaneous administration is evaluated on 30 guinea pigs in three groups (15 animals per group) by administering PEG-EPO and saline 0.5 ml subcutaneously at a dose of 1.5 and 15 μg / 250 g body weight of a guinea pig . Nonspecific inflammatory response to the administration of drugs is estimated from 24 hours to 7 days, inclusive.

The data obtained indicate that the drug "PEG-Erythropoietin" does not have an inflammatory effect after intracutaneous administration to guinea pigs and does not differ from the level of the inflammatory reaction in control animals, which were injected with saline. Thus, the study of the local action of the drug "PEG-Erythropoietin" after subcutaneous administration to guinea pigs showed the absence of any pathological changes at the injection site and regional lymph nodes.

The study of local action in intravenous administration of the drug to rabbits is carried out on 8 rabbits in two groups (4 animals per group) by administering PEG-EPO 1 ml to the marginal ear vein at a dose of 1.5 and 15 μg. Assessment of local action carried out on the basis of inspection data carried out within 7 days after priming of drugs, as well as the results of histological examination.

On the basis of the conducted studies, it was established that, when administered intravenously to rabbits, there are no pathological changes at the site of administration and regional lymph nodes.

B. Irritant effect.

The test for local irritating action is carried out according to the modified Dreis method on three rabbits. The drug "PEG-Erythropoietin" in a dose of 5 µg / 2.5 kg of body weight of the rabbit is applied to the right lower eyelid of the rabbit and keep the eyelids closed for 1 s. The reaction is recorded after 24, 48, 72 hours and on the 7th day. When recording the results of a conjunctival test in three rabbits after 24 hours, a brief expansion of some vessels of the conjunctiva was noted.

Taking into account the results of the experiments, the drug "PEG-EPO" when exposed to mucous membranes does not have an irritating effect.

Example 6. The study of the allergenic properties of the liquid pharmacological composition of the PEG-EPO.

A. Exploring immediate type hypersensitivity.

A study of immediate-type hypersensitivity is carried out on a model of guinea pigs weighing 250 g (240-260 g). To do this, the experimental group of 6 animals was administered PEG-EPO liquid pharmacological composition three times in 1 day at a dose of 2.5 µg / kg per injection (corresponds to one dose for a human): the first injection is subcutaneously, the next two are intramuscular. On the 21st day, after the first injection, a resolution dose equal to the total sensitizing dose of 7.5 µg / kg is injected intracardiacly. The control group (6 guinea pigs) according to the same scheme as the experimental group, injected with saline at 0.5 ml per injection. After the introduction of the resolving dose of the PEG-EPO liquid pharmacological composition in guinea pigs, no one type of immediate type hypersensitivity reaction was registered.

B. Study of delayed-type hypersensitivity.

A study of the allergenic effect of the delayed-type hypersensitivity is carried out on Ba1b / s mice (30 animals weighing 20-21 g, 2 experimental and 1 control group) in a plantar test under conditions of intensive sensitization of mice. The delayed-type hypersensitivity reaction index with the introduction of the PEG-EPO liquid pharmacological composition reaches 2.19, which indicates a weak irritant effect of the drug.

Based on the experiments, it was concluded that the liquid pharmaceutical composition PEG-EPO does not have an allergenic effect in the reaction of hypersensitivity of immediate and slow action, which confirms its immunological safety.

Example 7. The study of the immunogenic properties of the liquid pharmacological composition of the PEG-EPO.

Guinea pigs of both sexes weighing 240-260 g and Chinchilla rabbits of both sexes weighing 2.5 kg are used as biological models. Laboratory animals are divided into 10 groups: 4 experimental and 6 control.

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On the 56th and 92nd day of the study, animals from all experimental and control groups were sampled for blood. Serum is prepared from the blood by the usual method: blood samples are incubated for 45 minutes at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C. The resulting serum is centrifuged at 3500 rpm and stored at -65 ° C until the tests.

Antibody titer in sera obtained by immunization of animals with unmodified erythropoietin-beta and PEG-EPO liquid pharmacological composition is determined by direct enzyme-linked immunosorbent assay (TIFA). For this purpose, 0.1 ml of a solution of unmodified erythropoietin-beta at a concentration of 200 ng is introduced into the wells of the microplate. Antisera obtained from different groups of animals, contribute 0.1 ml to the wells in a series of serial dilutions twice. For the detection of the formed immune complex, anti-specific immunoglobulins conjugated with peroxidase are used (manufacturer — §1§ta). Measurements are carried out using a Vu-Kab Mobet tablet photometer 680.

In the conducted studies it was shown that some of the studied samples in various dosages possess immunogenic activity and stimulate the production of specific antibodies in guinea pigs and rabbits on the 56th and 92nd days.

The average titer of antisera after the introduction of unmodified EPO is in guinea pigs on the 56th day - 1/418, on the 92nd day - 1/580; in rabbits on the 56th day -1/670, on the 92nd day - 1/1050.

The average titer of antisera after administration of the PEG-EPO liquid pharmacological composition is 1/46 in guinea pigs on the 56th day, 1/92 on the 92nd day; in rabbits on the 56th day - 1/56, on the 92nd day - 1/106. Thus, the immunogenicity of the PEG-EPO liquid pharmacological composition is 8-10 times lower than that of unmodified EPO.

The results of the conducted studies prove that the developed liquid pharmaceutical composition of the drug "PEG-EPO" as part of the active active ingredient (PEG-EPO conjugate molecule) and auxiliary substances (stabilizers) is a low immunogenic drug.

Example 8. The study of the pharmacokinetic characteristics of the liquid pharmacological composition of the PEG-EPO.

A. Study on mice.

Prepared liquid pharmacological composition of PEG-EPO is estimated by the parameter of the prolongation of circulation in the blood of laboratory animals. As a comparison, use of the drug "Mircera" (manufacturer - R. Neutaii-La RosNe Ib., Series Н0481Н02), solution for intravenous and subcutaneous administration in a syringe-tube of 150 µg / 0.3 ml, valid until 02.2014.

As biological models, 96 CBA mice weighing 18–20 g, obtained from the nursery of the Russian Academy of Medical Sciences (p. Andreevka), are used. Animals are divided into three groups of 24 animals for each drug, one group of 4 animals is the control.

After distribution of laboratory animals into four groups of 24 animals for each drug and one control group of 4 animals, the samples under study are injected according to the following scheme: on the first day, 5 μg is administered subcutaneously to the interscapular region once a day (in terms of the erythropoietin protein content) 0.1 ml of a solution of the investigational drug "PEG-EPO", an unpegylated molecule of etropoetin and a comparison drug "Mircera". Next, blood is taken at 2, 6, 48, 72, 96, 120, 144, 168, 192, 216 and 240 hours after drug administration. In the control group, 0.1 ml of saline is administered.

The selection of blood for the study of pharmacokinetics carry out a simultaneous selection of blood from two mice at each point of the study. Serum is prepared from blood samples by the usual method: blood samples are incubated for 45 minutes at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the serum obtained is centrifuged at 3500 rpm and stored at -65 ° C until testing .

The concentration of erythropoietin in the blood serum is determined using the method of ELISA with the reagent kit "Erythropoietin-IFA-BEST" A-8776 (manufacturer - JSC "VectorBest", series No. 20) in accordance with the attached instructions. Measurements are carried out using a Vu-Kab Mobet tablet photometer 680.

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Table 8

The values of the concentrations of the studied drugs in the serum of mice after subcutaneous administration

According to the research results presented in table. 8, the following pharmacokinetic parameters are calculated using the Opsh 9.0.0 program (developed by OpttBa Sogr.):

T _max - the time to establish the maximum concentration;

C _max - the maximum concentration;

LIS _about-1 - area under the pharmacokinetic curve "concentration-time";

_B - volume of distribution;

T1 _{/ 2} - half-life;

С _max / ЛС _о-1 - relative rate of absorption;

СЛ - clearance,

the values of which are presented in table. 9.

Table 9

Values of pharmacokinetic parameters

Parameter The average value of the concentration of EPO in the blood, ng / ml "Mircera" PEG-EPO liquid pharmacological composition Erythropoietin Ttm, h 96 120 24 Stah. NG / ML 363 412 287 СЬ, ml / h 0.01 0.01 0.4 ν <|, ml 1.35 1.22 17.42 W / 2,4 101 100 29

The concentration of EPO in the blood of animals for the studied drugs is statistically significantly different, while the concentration and time profiles are generally similar. The composition according to the invention allows a slightly higher concentration of active substance to be created in the blood.

B. Study on rabbits.

In the experiments, chinchilla rabbits weighing 2.5-3.0 kg are used. The animals are divided into four groups: the first experimental group of 2 animals is subcutaneously injected in an amount of 3 μg / ml, the second - subcutaneously 10 μg / kg, the third - intravenously 3 μg / ml, the fourth group is the control.

Blood after administration of the drug is taken in the following time intervals: 0, 0.5, 24, 48, 72, 96, 120 and 144 hours. Blood is drawn from the ear vein in a volume of 2-3 ml for each test point.

Serum is prepared from blood samples by the usual method: blood samples are incubated for 45 minutes at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the serum obtained is centrifuged at 3500 rpm and stored at -65 ° C until testing .

The concentration of erythropoietin in serum is determined using the method of ELISA with the reagent kit "Erythropoietin-ELISA-BEST" A-8776 (manufacturer - JSC "Vector-Best", series No. 20) in accordance with the attached instructions. Measurements are carried out using a Vu-Kab Mobet tablet photometer 680.

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Table 10

The concentration values of the liquid pharmacological composition of PEG-EPO in the serum of rabbits

after subcutaneous and intravenous administration

Blood collection time, h The average value of the concentration of EPO in the blood, ng / ml. intravenously subcutaneously 3 mcg / kg 3 mcg / kg 10 mcg / kg 0 0 0 0 0.5 one - - 24 68.5 35.5 46.5 48 17 19.5 47.5 72 9 11.5 17 96 four 6 eleven 120 3 four 6.5

According to the research results presented in table. 10, the following pharmacokinetic parameters are calculated using the Advanced 9.0.0 program (developed by —Opt1–1aBaB):

T _max - the time to establish the maximum concentration;

C _max - the maximum concentration;

LIS _about-1 - area under the pharmacokinetic curve "concentration-time";

A _and - the volume of distribution;

T _1/2 - half-life;

С _max / ЛС _о-1 - relative rate of absorption;

SI - clearance

the values of which are presented in table. eleven.

Table 11

Values of pharmacokinetic parameters

Parameter The average value of the calculated pharmacokinetic parameters intravenously subcutaneously 3 mcg / kg 3 mcg / kg 10 mcg / kg Schschah, H 27.03 19.70 33,34 These *, NG / ML 105.57 36.02 52.17 Aiso-1, (ng-h) / ml 2160.67 1954.63 3192.08 WA, ml 28.42 83.29 191.68 1y, h 14.19 37.61 42.41 Stah / Aiso-ι, H ^-1 4.89 · 10 “ ^ζ ! _{> 84} . _10-a 1.63-kg ⁴ СЬ, ml / h 1.39 1.53 3.13

From the above calculation results, it is obvious that subcutaneous administration gives prolonged circulation of the drug in the blood.

Example 9. The preparation of a lyophilized pharmacological composition of PEG-INF.

Pre-prepare 5-fold concentrate of auxiliary components by dissolving in a volumetric flask with a capacity of 100 ml 20.0000 g of mannitol, 2.50 g of glycine, 0.75 g of I-methionine, 0.05 g of polysorbate 80.0.0375 g of EDTA and 3 , 90 g HaN ₂ PO ₄ -2H ₂ O in 90 ml of water and bring the final volume to the mark. Next, the resulting concentrate is filtered through a sterilizing filter with a pore size of 0.22 μm in sterile containers and stored at 2-8 ° C for no more than 5 days.

To prepare the pharmacological composition, 50 ml of PEG-INF solution with a concentration of 0.3 mg / ml and 35 ml of water are added to 10 ml of the concentrate, the pH is adjusted to 6.0 by adding 10% XaOH and the final volume is adjusted to 100 ml with water. The resulting solution is passed through a sterilizing filter with a pore size of 0.22 μm and poured under aseptic conditions of 1 ml in sterile vials. Next, the vials are loaded into a freeze dryer, frozen and dried under aseptic conditions under reduced pressure for 72 hours.

Example 10. The study of the stability of the lyophilized pharmacological composition of PEG-INF.

The vials are stored at 4 ° C, taking samples 1 time in 3 months. In each selected sample, specific activity, aggregate content, oligoform, pegylated and free INF and other indicators are determined according to the FSP specification. For this, the contents of each vial are reconstituted in 0.5 ml of water and the resulting solution is analyzed by size exclusion HPLC on a column 8reg4ex ™ 200 10/300 OI (10x300 mm, 13 μm) at room temperature and the flow rate of the mobile phase (0.15 M HaC1 in 50 mM phosphate buffer with pH 7) equal to 0.5 ml / min. Detection is carried out spectrophotometrically at 280 nm. The results of the analyzes are presented in table. 12.

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As can be seen from the table. 12, the lyophilized PEG-INFP pharmacological composition sealed in dark glass vials of type 1 (EIG. PE, IPZ), meet the requirements of the draft FSP on the parameter stability for 2 years and 3 months when stored at 2 to 8 ° C the place protected from light.

Table 12

Analytical data confirming the stability of the lyophilized pharmaceutical composition PEG-IFN with the studied shelf life

Description White powder that does not contain impurities. Hygroscopic. Authenticity BUT Must have specific activity. B The retention time of the main peak in the chromatogram of the test sample corresponds to the retention time of the main peak in the chromatogram of the standard sample. Time of dissolution Must dissolve within 3 minutes. Transparency of the reconstituted solution Dissolved drug must withstand comparison with reference I. Chroma recovered solution Dissolved drug must withstand comparison with standard Υ. pH 5.5 to 6.5 Mechanical inclusions Must meet the requirements for the determination of visible particles. Loss on drying Not more than 10%. Precision filling The coefficient of variation for dry matter is not more than 10% Sterility Must be sterile. Bacterial endotoxins Not more than 25 ES in 0.5 ml. Abnormal toxicity Must be non-toxic. quantitation From 85 to 115% of the stated amount. Purity Not less than 85%. Foreign compounds (related compounds) Not more than 10% poly peginterferon alpha-2b and not more than 5% interferon alpha-2b. Specific activity From 1 · 10 ⁷ to 3 · 10 'IU / mg protein.

Batch number, dosage | Inspection date Description Authenticity Time of dissolution min Transparency restored solution Chromaticity restored solution £ Mechanical inclusions Weight loss drying Precision filling one { Bacterial endotoxins Abnormal Toxicity Quantitative definition I one Multilateral connections (related connections) Specific activity BUT B 07.09 Corresponding to 13 6.30 5.5 3 98 92.4 6.6 + 1 2.7-10 ⁷ 10.09 Corresponding to 1.5 6.30 5.0 2 98 92.6 6.1 + 13 2.7-10 ⁷ 01.10 Corresponding to 13 635 63 3 § •P about g but "L οί 98 91.7 6.1 + 1.2 2.5-10 ⁷ & 04.10 & to Corresponding to 13 B & 6.33 dregs 53 2 £ 97 913 6.2 + 2.3 2,5-Ю ⁷ eight 07.10 Corresponding to 13 § 633 5.6 2 eight § 97 91.4 6.2 + 2.4 2,3-10 ’ 010709, 10.10 | one Corresponding to 13 one 6.30 5.4 2 | 97 91.7 63 + 3.0 2,2-Ю ⁷ 01.11 δ δ Corresponding to 13 δ δ 6.30 6.0 2 about " δ 97 90,8 5.9 + 3.3 23S ⁷ 04.11 Corresponding to 1.5 6.25 5.3 3 § 97 90.1 5.6 + 3.3 23S ⁷ 07.11 Corresponding to 13 6.28 5.5 3 97 90.0 53 + 4.7 23S ⁷ 10.11 Corresponding to 13 6.30 6.0 3 96 89.9 5.3 + 4.8 2.1-10 ’ 07.09 Corresponding to 2 6.10 5.8 2 101 96 2.2 + 1.8 23S ⁷ 10.09 Corresponding to 2 6.12 5.7 2 98 96.4 23 + 1.8 2,4-10 ’ 01.10 Corresponding to 2 6.05 5.8 3 one about 98 96.2 2.3 + 13 2,4-10 ’ and 04.10 £ £ Corresponding to 2 one £ 6.00 5.9 2 δ 98 95.9 2.0 + 2.1 2,4-10 ’ 3 07.10 Corresponding to 2 uglazh 6.00 5.8 2 δ § I but <h 98 95,8 1.9 + 2.3 23S ⁷ ί 10.10 § yo Corresponding to 2 one 5.95 one 53 2 one | 98 95.4 1.8 + 2.8 2,3-10 ’ 3 about 01.11 δ δ Corresponding to 2 δ δ 5.90 δ 5.4 2 Less than 1 δ 98 95.1 1.8 + 3.1 2,3-10 ’ 04.11 Corresponding to 2 5.90 5.7 3 98 94.5 1,544.0 2,3-10 ’ 07.11 Corresponding to 2 5.92 5.6 3 97 943 1.6 + 3.9 2,3-10 ’ 10.11 Corresponding to 2 5.95 5.6 3 97 94.3 1,244,5 2,2-10 ’

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030709.50 mcg 07.09 Complies Complies So. 1.5 Complies Complies 6.25 Complies 24 2 [Sterilen | Less than 12.5 EU per 0.5 ml Complies 95 98.5 1.0 + 04 2410 ⁷ 10.09 So. 14 6.20 2.4 2 95 98.4 1.1 + 0.5 24 th ⁷ 01.10 Corresponding to 14 6.23 2.5 2 95 97.6 1.0 + 1.4 2.5-10 ⁷ 04.10 Corresponding to 1.5 6.15 2.6 2 94 97,8 1.1 + 1.1 2.5-10 ⁷ 07.10 Corresponding to 14 6.10 2.8 3 94 974 1.0 + 14 2.5-10 ⁷ 10.10 Corresponding to 14 6.15 2.0 2 94 974 1.1 + 1.4 2.4-10 ⁷ 01.11 Corresponding to 14 6.10 24 2 91 97.3 1.0 + 1.7 2.4-10 ⁷ 04.11 Corresponding to 14 6.05 2.7 2 91 96.2 0.9 + 2.9 2.4-10 ⁷ 07.11 Corresponding to 14 6.10 2.1 2 91 96.4 0.8 + 2.8 2.4-10 ⁷ 10.11 Corresponding to 14 6.05 2.0 2 90 96.0 0.5 + 34 2.4-10 ⁷ 07.09 Corresponding to 14 6.40 4.5 3 103 98.1 14 + 0.4 2.7-10 ⁷ 10.09 Corresponding to 14 6.38 4.0 2 101 97,8 1.4 + 0.8 2.5-10 ⁷ 01.10 Corresponding to 14 6.30 4.0 four § 100 97.6 1.4 + 1.0 2.5-10 ⁷ 04.10 δ δ Corresponding to 14 δ 6.31 δ 4.2 3 about δ 100 97.4 14 + 1.4 2.6-10 ⁷ £ § 07.10 Corresponding to 14 645 4.1 2 at ABOUT F 2 >. 100 97,8 1.1 + 1.1 2.6-10 ⁷ ί 10.10 rz Corresponding to 1.5 | with δ 6.30 Β 4.2 3 δ og 98 964 1.1 + 2.7 24 th ⁷ one 01.11 δ δ Corresponding to 14 about ABOUT ο υ 6.28 δ 4.0 2 eight about ABOUT 98 96.1 1.0 + 2.9 2.5-10 ⁷ 04.11 Corresponding to 14 6.25 4.0 2 F 2 99 95.5 0.8 + 3.7 2.5-10 ⁷ 07.11 Corresponding to 14 6.25 4.4 3 98 95.1 0.6 + 4.3 2.4-10 ⁷ 10.11 Corresponding to 14 6.28 44 3 98 94.9 0.6 + 44 2.4-10 ⁷ 07.09 Corresponding to 14 6.22 5.1 3 97 97.5 2.4 + 0.1 2.8-10 ⁷ 10.09 Corresponding to 14 6.25 5.2 2 97 97.4 2.3 + 0.3 2.8-10 ⁷ 01.10 Corresponding to 14 6.20 5.0 3 10.5 ml 95 97.2 2.1 + 0.7 2.9-10 ⁷ and g * 04.10 δ δ Corresponding to 1.5 δ δ 6.20 δ 4.9 3 δ 96 96.6 2.0 + 1.4 2.7-10 ⁷ £ L * & δ I § 07.10 £ δ Corresponding to 14 δ 6.15 4.8 3 2 B 94 97.0 2.1 + 0.9 2.7-10 ⁷ one 10.10 £ Corresponding to 14 one 6.19 Β 5.0 2 "about οί 94 96.9 2.0 + 1.1 24 th ⁷ about 01.11 δ δ Corresponding to 14 δ δ 6.20 (§ 5.2 3 and 92 96.3 1.9 + 1.8 2.5-10 ⁷ 04.11 Corresponding to 1.5 6.05 4.9 3 one 91 95,8 1.8 + 2.4 2,5-Ю ⁷ 07.11 Corresponding to 14 6.08 4.8 2 90 95.4 1.7 + 2.9 2.4-10 ⁷ 10.11 Corresponding to 14 6.00 5.0 3 90 95.5 1.6 + 2.9 2.4-10 ⁷ 07.09 Corresponding to 2 6.10 4.2 2 94 96.1 3.5 + 0.4 2.8-10 ⁷ 10.09 Corresponding to 2 6.10 4.1 2 94 96.0 3.4 + 0.6 2.7-10 ⁷ 01.10 Corresponding to 2 6.08 4.1 2 § 95 95,8 3.3 + 0.9 2.7-10 ⁷ B 04.10 δ δ Corresponding to 2 δ δ 6.12 δ 4.0 2 about' δ 93 95.7 3.1 + 14 2.7-10 ⁷ £ > » >. & X Well >> £ 07.10 B $ Corresponding to 2 five Ι 6.05 <3 4.5 2 2 93 95.4 3.1 + 14 2.7-10 ⁷ one 10.10 £ Corresponding to 2 one s β 6.02 Β 3.8 2 I 3 £ 92 95.1 3.2 + 1.7 2.7-10 ⁷ ξ about 01.11 δ δ Corresponding to 2 δ δ 6.00 δ 4.2 2 ϊ δ 92 94.6 3.0 + 2.3 2.6-10 ⁷ 04.11 Corresponding to 2 6.00 3.9 2 92 944 24 + 3.3 2.6-10 ⁷ 07.11 Corresponding to 2 5.95 4.0 3 91 94.3 2.1 + 3.6 2.6-10 ⁷ 10.11 Corresponding to 2 5.80 4.5 2 89 94.1 1.9 + 4.0 2.6-10 ⁷ 07.09 Corresponding to 14 6.41 7.0 2 98 95.5 4.2 + 0.3 2.5-10 ⁷ 10.09 Corresponding to 14 6.39 64 3 97 95,8 4.0 + 0.2 2.5-10 ⁷ 01.10 Corresponding to 14 6.45 64 four § 98 95.4 3.9 + 0.7 2.5-10 ⁷ ι mcg 04.10 one δ Corresponding to 14 one one 6.38 is growing 6.0 3 X 2 St. F δ 97 954 3.9 + 0.8 2.5-10 ⁷ § 07.10 £ Corresponding to 14 0 647 6.2 2 about 2 five 96 95.0 3.8 + 14 2.4-10 ⁷ ί 10.10 § Corresponding to 14 one one 645 Sootve 6.0 3 δ sch 95 94.8 3.6 + 1.6 2.4-10 ⁷ £ about 01.11 δ δ Corresponding to 14 δ δ 648 64 2 eight δ 94 94.7 3.2 + 2.1 2.4-10 ⁷ 04.11 Corresponding to 14 640 6.0 3 one 94 94.4 3.0 + 2.6 2.3-10 ⁷ 07.11 Corresponding to 14 6.25 6.1 3 94 94.2 2.9 + 2.9 2.3-10 ⁷ 10.11 Corresponding to 14 6.28 6.0 2 92 93,8 2.5 + 3.7 2.3-10 ⁷

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141009,150 mcg 07.09 Th Complies Corresponding to one Complies Complies 5.95 Complies 33 3 | Sterilen | Less than 12.5 EU per 0.5 ml Complies 96 98.1 1.7 + 0.2 2.5-10 ⁷ 10.09 Corresponding to one 5.90 3.2 3 95 97.5 1.6 + 0.9 2.5-10 ⁷ 01.10 Corresponding to one 5.98 3.3 3 95 97.3 1.5 + -1.2 23-10 ⁷ 04.10 Corresponding to one 6.00 3.0 3 94 97.4 1.4 + -1.2 2.5-10 ⁷ 07.10 Corresponding to one 5.95 ZZ 3 95 97.1 1.2 + -1.7 23 · Yu ⁷ 10.10 Corresponding to one 5.98 33 3 93 96.5 1.1 + -2.4 2.5-10 ⁷ 01.11 Corresponding to one 5.95 3.0 3 93 95.9 1.0 + 3.1 2.4-10 ⁷ 04.11 Corresponding to one 5.90 3.1 3 93 95.4 1.0 + 3.6 2.4-10 ⁷ 07.11 Corresponding to one 5.98 3.2 3 92 953 0.9 + 3.6 2.4-10 ⁷ 10.11 Corresponding to one 5.95 3.2 3 92 95.0 0.9 + 4.1 2.4-10 ⁷ 151009,150 mcg 07.09 Complies Complies Corresponding to 1L Complies Complies 6.30 Complies 23 3 | Sterilen | Less than 12.5 EU per 0.5 ml Complies 98 98.8 0.9 + 0.3 2.6-10 ⁷ 10.09 Corresponding to 1.5 6.26 2.4 3 98 98.5 0.9 + 0.6 2.6-10 ⁷ 01.10 Corresponding to 13 6.35 23 3 97 98.4 0.8 + 0.8 2.7-10 ⁷ 04.10 Corresponding to 1.5 6.28 2.6 3 97 98.1 0.8 + 1.1 2.6-10 ⁷ 07.10 Corresponding to 1.5 6.12 2.0 four 97 97.9 0.8 + 1.3 2.6-10 ⁷ 10.10 Corresponding to 13 6.10 2.1 3 96 97.6 0.7 + 1.7 2.6-10 ⁷ 01.11 Corresponding to 1.5 6.13 2.2 3 96 97.4 0.6 + 2.0 2.5-10 ⁷ 04.11 Corresponding to 1.5 6.20 23 3 96 97.3 0.5 + 2.2 2.5-10 ⁷ 07.11 Corresponding to 1.5 6.05 2.6 2 96 97.1 0.4 + 2.5 23 · Yu ⁷ 10.11 Corresponding to 1.5 6.25 2.8 2 95 96.5 0.3 + 3.2 23 · Yu ⁷ 161009,150 mcg 07.09 Complies Complies Corresponding to 13 Complies Complies 6.10 Complies 3.0 four | Sterilen | Less than 12.5 EU per 0.5 ml Complies 95 98.9 0.9 + 0.2 2.6-10 ⁷ 10.09 Corresponding to 1.5 6.10 3.1 2 95 98.7 0.9 + 0.4 2.6-10 ⁷ 01.10 Corresponding to 13 6.05 3.2 four 95 98.6 0.8 + 0.6 2.6 · 10 ⁷ 04.10 Corresponding to 13 6.08 3.5 2 94 98.4 0.7 + 0.9 2,5 · Ю ⁷ 07.10 Corresponding to 13 6.02 2.9 four 93 98.0 0.7 + 13 23 · Yu ⁷ 10.10 Corresponding to 13 5.99 3.0 four 93 97.6 0.6 + 1.8 2,5 · Ю ⁷ 01.11 Corresponding to 13 5.99 3.0 3 92 97.1 0.5 + 2.4 2.4-10 ⁷ 04.11 Corresponding to 13 5.95 2.9 3 92 96,8 0.5 + 2.7 2.4-10 ⁷ 07.11 Corresponding to 13 5.90 2.5 3 92 95.9 0.4 + 3.7 2.4-10 ⁷ 10.11 Corresponding to 13 5.91 3.0 2 89 95.5 0.4 + 4.1 2.3-10 ⁷

Example 11. The determination of the specific activity of the lyophilized pharmacological composition of PEG-INF.

The antiviral specific activity (CA) of unmodified interferon a-2b and the pharmaceutical composition containing the monoPEG-ΙΝΡ a-2b conjugate was determined by ίη νίίΐΌ in cell line culture ΜΏΒΚ (manufacturer - "Biolot"), sensitive to alpha-interferon, obtained on 10- 28 passageway infected with vesicular stomatitis virus.

The results of a comparative study with the use of commercial pegylated interferon “Pegasys” and “PegIntron” and the international standard sample of unmodified interferon are presented in Table. 13.

Table 13

The results of the comparison of the specific activity of various drugs made on the basis of pegylated interferon

A drug CA sample, IU / mg protein % CA from native interferon ΙΝΡ a-2b 200 * 10 ^b 100 The drug PEG-IFN alpha-2b * 64 * 10 ^b 32 PegIntron 52 * 10 ^b 26 Pegasys 2 * 10 ^b one

* Prepared in accordance with the invention.

The size and structure of polyethylene glycol attached to the interferon slightly reduces the antiviral activity due to the screening of the active sites of the INF.

In the next series of experiments, it was found that the values of CA in the solution and in the lyophilisate did not differ significantly and, after storage for 42 months, the specific activity of the lyophilisate did not change (Table 14). The specific activity of the finished dosage form of the "PEG-interferon alpha-2b" preparation prepared in accordance with the invention is comparable to a specific 19 029942

physical activity of the drug "PegIntron" (23-28%) and significantly exceeds that of the drug "Pegasys" (0.5-3%).

Table 14

The results of the study of the specific activity of the drug "PEG-interferon alpha a-2"

A drug Specific activity, IU / mg ΙΝΡα-26 200-10® PEG-ΙΝΡ a-2b before making stabilizer 64-10® PEG-ΙΝΡ a-2b after making stabilizer before lyophilization 64-10® PEG-ΙΝΡ and-2b after lyophilization 69-10® PEG-ΙΝΡ a-2b (series ΡΑΙ493 е§1.) After lyophilization and 42 months of storage 62-10®

Example 12. Preclinical study of the toxic effects of the lyophilized pharmacological composition PEG-IFN.

A. Determination of the acute toxicity of the lyophilized pharmacological composition PEGINP.

The study of acute toxicity is carried out on mice (50 animals) and guinea pigs (50 animals) in five experimental groups of each animal species, 5 males and 5 females in each group. Animals of the 1st group injected solvent (water for injection). Animals of the 2nd and 4th groups are injected with the PEG-INFA pharmacological composition under test, animals of the 3rd and 5th groups receive the PegIntron comparison preparation (manufacturer - 8Bliepp-P1oiG). The study uses therapeutic and tenfold therapeutic doses for humans.

On the 15th day of the study, animals are subjected to euthanasia with the inspection of macrodamages of organs, and samples are taken for hematological, biochemical, and histological examination. The use of the PEG-IFN pharmacological composition and the PegIntron comparison drug in the study of acute toxicity does not cause animal death, does not lead to weight loss, the formation of inflammatory changes, the development of necrobiotic processes at the injection site, which indicates the absence of intoxication symptoms.

B. Determination of chronic toxicity of the lyophilized pharmacological composition of PEG-INF.

Chronic toxicity studies are conducted on mice (100 animals) and guinea pigs (100 animals) in five experimental groups of each species, 10 males and 10 females in each group. Animals of the 1st group injected solvent (water for injection). Animals of the 2nd and 4th groups are administered the PEG-IFN test pharmacological composition, animals of the 3rd and 5th groups receive the PegIntron comparison drug. The drugs are administered within 28 days. The study uses therapeutic and tenfold therapeutic doses for humans.

On the 29th and 43rd day of the study, animals are subjected to euthanasia with an inspection of macrodamages of organs, and samples are taken for hematological, biochemical, and histological examination. During the study, no death of animals decreased motor and nutritional activity, changes in physiological functions, there is no decrease in body weight and internal organs, which indicates the absence of a toxic effect of the drug on the systems and organs of laboratory animals.

At planned necropsy, pathological and histological studies performed show that a single and repeated administration of PEG-Interferon and PegIntron in doses of 1.5 and 15 μg / kg does not cause the development of pathological, including inflammatory, dystrophic and necrotic processes in internal organs of animals. The results of the analysis of hematological and biochemical parameters of the blood of laboratory animals in the "acute" and "chronic" experience show the absence of pronounced pathological changes in the indicators of general and biochemical blood analysis, as well as the absence of significant differences between the experimental and control groups of animals.

The pharmacological composition of PEG-INF is pyrogen-free, since intravenous administration to rabbits at a dose of 15 μg / 2.5 kg, the sum of the temperature increases does not exceed 2.2 ° C.

Example 13. Determination of the local and local irritating action of the lyophilized pharmacological composition of PEG-INFF after subcutaneous administration.

Local action is assessed on 8 guinea pigs (4 animals per group) by administering the PEG-IFN pharmacological composition and saline 0.5 ml subcutaneously at a dose of 15 μg / 250 g body weight. Nonspecific inflammatory response to the administration of drugs is estimated from 24 hours to 5 days inclusive.

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After intradermal administration to guinea pigs, the level of inflammatory response in animals in the experimental and control groups is not significantly different. This indicates that the pharmacological composition of PEG-INF does not exhibit an inflammatory effect.

When assessing the local action of the pharmacological composition of PEG-INF in guinea pigs, there are no pathological changes at the site of administration and regional lymph nodes.

The test for local irritating action for eyes is carried out according to the modified Dreis method on 6 rabbits. The pharmaceutical composition of PEG-IFN in a dose of 15 µg / 2.5 kg of body weight of the rabbit is applied to the right lower eyelid and keep the eyelids closed for 1 s. The reaction is recorded after 24, 48, 72 hours and on the 7th day. When recording the results of a conjunctival test in 2 rabbits after 24 hours, a short-term dilation of some vessels of the conjunctiva was noted. From the results it follows that the drug "PEG-Interferon" when exposed to the mucous membranes does not have an irritating effect.

Example 14. The study of the allergenic properties of the lyophilized pharmacological composition of PEG-IFN.

A. The study of the hypersensitivity of the immediate type of the lyophilized pharmacological composition of PEG-INF.

A study of immediate-type hypersensitivity is carried out on a model of guinea pigs weighing 250 g (240-260 g). Experimental animals (6 guinea pigs) were administered PEG-INFP pharmacological composition three times in 1 day at a dose of 1.5 µg / kg (1 human dose) per injection: the first injection was subcutaneously, the next two were intramuscular. On day 21 after the first injection, a resolution dose equal to a total sensitizing dose of 4.5 µg / kg is administered intracardiacly. Animals of the control group (6 guinea pigs) according to the same scheme as in the experimental group, injected with saline 0.5 ml. After the introduction of the resolving dose in animals sensitized with the pharmacological composition of PEG-INFA, no type of immediate type hypersensitivity reaction was registered in guinea pigs.

B. Study of delayed-type hypersensitivity of the lyophilized PEG-IFN pharmacological composition.

A study of the allergenic effect of the delayed-type hypersensitivity is carried out on Ba1b / C mice weighing 20-21 g (20 mice) in plantar test under conditions of intensive sensitization of mice. The index of the reaction of hypersensitivity of the delayed type with the introduction of the pharmacological composition PEG-INF reaches 2.17, which indicates a weak irritant effect of the drug.

Based on the experiments, it was concluded that the lyophilized pharmacological composition PEG-INFA does not have an allergenic effect in the immediate and delayed hypersensitivity reactions, which confirms its immunological safety.

Example 15. The study of the immunogenic properties of the lyophilized pharmacological composition of PEG-IFN.

As biological models, 20 Bál / C mice weighing 18–20 g, obtained from the nursery of the Russian Academy of Medical Sciences (p. Stolbovaya), are used. Animals are divided into 4 groups of 5 animals (two experimental and two control groups).

Mice of the first experimental group were administered, once a week for 5 weeks, 1.0 μg of the drug of the PEG-interferon alpha-2b conjugate prepared in accordance with the invention in a volume of 0.1 ml subcutaneously into the interscapular region in a volume of 0.1 ml. Mice of the second experimental group were administered subcutaneously 5.0 μg of the same preparation in the amount of 0.1 ml subcutaneously into the interscapular area once a week for 5 weeks.

Mice of the first control group were administered 1 million IU of unmodified interferon alfa-2b in the volume of 0.1 ml subcutaneously to the interscapular area once a week for 5 weeks. Mice of the second control group of animals injected with 0.1 ml of saline.

At the end of the fifth week, all mice in the group are given a one-time blood draw. Serum is prepared from the blood by the usual method: blood samples are incubated for 45 minutes at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the serum obtained is centrifuged at 3500 rpm and stored at -65 ° C until the tests are performed.

Antibody titers in sera obtained by immunization of mice with unmodified IFN and PEG-IFN conjugates are determined using direct enzyme-linked immunosorbent assay (TYPE). For this purpose, 0.1 ml of unmodified interferon alfa-2b solution at a concentration of 200 ng is introduced into the microplate wells. Antisera obtained from different groups of animals, contribute 0.1 ml to the wells in a series of serial dilutions twice. To detect the resulting immune complex, anti-mouse immunoglobulins conjugated with peroxidase are used. Measurements are carried out using a plate photometer Βίο-Kai Mobe1 680.

The titer of the antiserum after administration of unmodified IFN is 1/1024. The titer of antisera after the introduction of the conjugate PEG-IFN at a concentration of 1.0 μg / mouse is 1/64, and after the introduction of the conjugate PEG-IFN at a concentration of 5.0 μg / mouse is 1/128. The results of the study are presented in Table. 15.

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Table 15

The results of the analysis of antisera after the introduction of the lyophilisate PEG-IFN

Sample name Content in OD ml Antiserum titer Control 0 0 Unmodified IFN alpha 2b 1 million IU 1/1024 PEG-IFN Lyophilisate 1.0 mcg 1/64 PEG-IFN Lyophilisate 5.0 mcg 1/64

Thus, from the obtained results it follows that the immunogenicity of the studied lyophilized pharmacological composition of PEG-IFN is 16 times lower than that of unmodified IFN.

Example 16. The study of the pharmacokinetic characteristics of the lyophilized pharmacological composition of PEG-IFN.

The prepared lyophilized pharmacological composition of PEG-IFN was evaluated by the parameter of circulation prolongation in the blood of laboratory animals. As a comparison drug, PegIntron (manufacturer — ZeBePG-P1oiG) is used, a lyophilisate for preparing a solution for subcutaneous administration in 80 μg syringe pens, series 9-ΙΚΟ-60127, valid until 10.2012.

As biological models, 150 CBA mice weighing 18–20 g, obtained from the nursery of the Russian Academy of Medical Sciences (p. Stolbovaya) are used. Animals are divided into 12 groups of 5 animals for each drug, two groups of 15 animals are the control.

On the first day, the mice are injected subcutaneously once in the interscapular area with 1 μg in 0.1 ml of the PEG-IFN preparation solution prepared in accordance with the invention and the PegIntron reference preparation in terms of the protein (interferon) content. Then blood is taken 2, 4, 6, 12, 18, 24, 48, 72, 96, 120 and 144 hours after the administration of drugs. One control group is administered 0.1 ml of saline, the other control group consists of intact animals.

The selection of blood for the study of pharmacokinetics is carried out by one-time decapitation of 3-5 mice at each point of the study. Serum is prepared from the blood by the usual method: blood samples are incubated for 45 minutes at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the serum obtained is centrifuged at 3500 rpm and stored at -65 ° C until the tests are performed.

The concentration of pegylated interferon in serum is determined simultaneously in all samples using the enzyme immunoassay method with the Alpha-Interferon-IFA-BEST reagent kit A-8755 (manufacturer: ZAO Vector-Best, series No. 20) in accordance with the attached instruction. Measurements are carried out using a Vu-Kai-Moye1 680 tablet photometer. Based on the results, pharmacokinetic parameters are calculated (Table 16).

Table 16

Values of pharmacokinetic parameters

Parameter A drug The composition of the PEG-IFN PegIntron Schschah, H 17.3 9.4 Stach, NG / ML 166 thirty WA, ml 6.0 33.3 ίιζζ, h 22.2 11.8 Stakh / AiSo-Ch ' ¹ 0.031 0.059 СЬ, ml / h 0.19 1.96

From the analysis of the averaged pharmacokinetic data of the studied drugs it can be seen that their profiles do not match. From the above values it can be seen that the lyophilized pharmacological composition of PEG-IFN in terms of circulation time and the concentration created in the blood at an equal dosage greatly exceeds the commercially produced drug "PegIntron".

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Claims (5)

CLAIM 1. A pharmaceutical composition presented in a liquid or lyophilized finished dosage form (SFF) containing pegylated protein, characterized in that the pegylated protein, in which polyethylene glycol fragments are covalently attached to the protein molecule by means of azo groups, corresponds to formula (I) where rgo! - biologically active protein selected from interferon and erythropoietin; A - amino acid fragment of histidine; B - amino acid fragment of tyrosine; n is an integer in the range from 10 to 1200; t - takes values from 0 to 10; k - takes values from 0 to 10, and m ^ k, if m or k is 0, in the form of a conjugate or mixture of conjugates with different values of n, m and k, as well as pharmacologically acceptable auxiliary ingredients. 2. The pharmaceutical composition according to claim 1, wherein the pharmacologically acceptable auxiliary ingredients are selected from cosolvents, salts, acids, bases, antioxidants, preservatives, amino acids, proteins, carbohydrates, polymers, detergents and chelating agents. 3. The pharmaceutical composition according to claim 1 or 2, characterized in that the biologically active protein is erythropoietin and the composition is presented in the form of liquid SFF. 4. The pharmaceutical composition according to claim 1 or 2, characterized in that the biologically active protein is an interferon and the composition is presented in the form of lyophilized SFF. 5. The pharmaceutical composition according to claim 2, characterized in that it contains from 0.01 to 50 mg of a conjugate of formula (I), contains 5 to 100 mg of mannitol as a carbohydrate, contains 0.1 to 20 mg of glycine as an amino acid It contains from 0.1 to 10 mg of L-methionine as an antioxidant, from 0.01 to 1 mg of polysorbate 80 as a detergent, contains from 0.01 to 1 mg of ethylenediaminetetraacetic acid as a complexone, and from 1 up to 25 mg of sodium dihydrogen phosphate in terms of anhydrous salt, contains sodium hydroxide as a base, as acid contains acetic or hydrochloric acid in an amount that provides a pH of 3.5 to 7.0; as a solvent it contains water for injection up to 1 ml.