Novel pharmaceutical composition

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a liquid pharmaceutical composition comprising pegilated erythropoietin as a conjugate in pharmaceutically acceptable buffer at pH from 5.5 to 7.0 and optionally one or some pharmaceutically acceptable excipients. Proposed composition is used in treatment and prophylaxis of diseases associated with erythropoiesis injury. The advantage of invention involves enhancing stability of the preparation.

EFFECT: improved and valuable property of preparation.

59 cl, 4 tbl, 11 dwg, 13 ex

 

The present invention relates to a liquid pharmaceutical composition containing the protein erythropoietin, multiply charged inorganic anion in a pharmaceutically acceptable buffer suitable for maintaining the pH of the solution in the range from approximately 5.5 to approximately 7,0, and optionally one or more pharmaceutically acceptable excipients. Such a composition is preferably used for the prevention and treatment of diseases associated with erythropoiesis.

Erythropoiesis is the process of formation of red blood cells, which serves to replenish cellular destruction. Erythropoiesis is a controlled physiological mechanism, resulting in the formation of the number of red blood cells, sufficient for the corresponding saturation of the tissues with oxygen. Naturally occurring human erythropoietin (Nero) is produced in the kidney and is a humoral plasmatic factor that stimulates the formation of red blood cells (.Carnot and .Deflandre, C.R.Acad. Sci. 143: 432 (1906); A.J.Erslev, Blood 8: 349 (1953); K.R.Reissmann, Blood 5: 372 (1950); L.O.Jacobson, E.Goldwasser, W.Freid and L.F.Pizak, Nature 179: 6331-6334 (1957)). Naturally occurring EPO stimulates the division and differentiation commiteeman erythroid precursors in the bone marrow and exerts its biological activity by binding the receptors on retainig predecessors (B.S.Krantz, Blood 77: 419 (1991)).

Erythropoietin was obtained by biological synthesis using techniques of recombinant DNA (J.C.Egrie, T.W.Strickland, J.Lane, etc., Immunobiol. 72: 213-224 (1986)), and it represents the product of the gene cloned human EPO, which was integrated and expressed in tissue cells of the Chinese hamster ovary (Cho-cells). Primary structure of the dominant fully protestirovanny forms Nero presented in SEQ ID NO:1. It contains two disulfide bridge between Cys7-Cys161and Cys29-Cys33. The molecular mass of the polypeptide chain of EPO without fragments sugar is 18236 Yes. In the intact molecule EPO approximately 40% of the molecular weight accounted for carbohydrate groups, which lead to glycosylation of the protein at the sites of glycosylation of the protein (H.Sasaki, .Bothner, A.Dell and .Fukuda, J.Biol. Chem. 262: 12059 (1987)).

Because human erythropoietin plays a key role in the formation of red blood cells, this hormone can be used in the treatment of blood disorders characterized by low production of red blood cells or the production of abnormal red blood cells. In clinical conditions, EPO is used to treat anemia in patients with chronic renal failure (CRF) (J.W.Eschbach, J.C.Egri, M.R.Downing and others, NEJM 316: 73-78 (1987); J.W.Eschbach, M.H.Abdulhadi, J.K.Browne, etc.. Ann. Intern. Med.111: 992 (1988); J.C. Egrie, J.W. Eschbach, T.McGuire, J.W.Adamson, Kidney Intl. 33: 262 (188); V.S.Lim, R.L.Degowin, D.Zavala, etc.. Ann. Intern. Med. 110: p.108-114 (1989)) and patients suffering from AIDS, and patients suffering from cancer and undergoing chemotherapy (R.P.Danna, S.A.Rudnick, R.I. Abels in: Garnick M.B. (editor) Erythropoietin in Clinical Application - An International Perspective, New York, NY: Marcel Dekker; str-324 (1990)).

Known pharmaceutical compositions have at least one of the following drawbacks:

They are lyophilizate. Besides the fact that the production process is complicated by the lack of liofilizatow is that you must restore them before carrying out the injection of the people. This leads to the need for medical personnel additional operations that is difficult and fraught with risk of improper handling of the pharmaceutical product;

They contain additives as human serum albumin. Because the human serum albumin is a product obtained from the total water of the human body, there is a risk of viral infections in the result of contamination of the albumen of the drug. Possible allergic reactions.

- All applicants currently on sale composition containing erythropoietin, are unstable at elevated temperatures, i.e. at temperatures higher than the temperature of the refrigerator, which, as a rule, is 2-8°C. Therefore, they must be stored in the refrigerator (2-8° (C) and cannot be stored at room temperature (approximately 20°). This leads to additional costs associated with storage at low temperatures and the need to have for this equipment, and also makes use of the medication. The concept of "unstable" in the context of the present description indicates that the storage at elevated temperatures, for example, at 25°With over a sustained period of time (e.g. several months or more than 6 months) leads to the decomposition of protein. In the context of the present description the term "biodegradable" refers to physical changes (e.g., aggregation or denaturation) and chemical changes (for example, oxidation or, in General, modification of chemical bonds) protein, which are known to occur primarily at elevated temperatures (above 8°). Incubation of the protein at temperatures close to or above its transition temperature (also known as the melting temperature), leads to the deployment of the protein, i.e. losing the native structure and biological activity of the polypeptide. The temperature of the phase transition is strictly correlated with the stability of the protein and dependence is from the environment, where is the protein (e.g., pH, presence of salts, ionic strength, presence of a buffer substance and the like). For example, denaturation can lead to aggregation of the molecules of erythropoietin, i.e. the formation of dimers (covalent or non-covalent bonds, units of a higher order and even individual particles. This leads to reduced efficacy of a drug and can cause unwanted side effects after the injection of the people.

Thus, the present invention was based on the task of creating a pharmaceutical composition, which allows to minimize or eliminate the above disadvantages.

According to the present invention the task is solved by creating a pharmaceutical composition containing the protein erythropoietin, multiply charged inorganic anion in a pharmaceutically acceptable buffer solution having a pH value in the range from approximately 5.5 to approximately 7,0, and optionally one or more pharmaceutically acceptable excipients.

With the invention it has been unexpectedly found that the inclusion of erythropoietin in this composition improves its stability at temperatures higher than the temperature of the refrigerator (2-8°C), especially at room temperature (i.e. below 25°), and even at higher temperatures is x, for example, if 40°C. This means that the composition can be stored without refrigeration for an extended period of time and there is no significant loss of activity and significant decomposition.

Unless otherwise stated, the terms used to illustrate and define the meaning and scope of various terms used in the description of the present invention, have the following meanings.

The notion of multiply charged inorganic anion" refers to an inorganic anion having two or more negative charges on the molecule, for example, anion sulfate SO42-or phosphate anion, i.e. acidic phosphate HPO42-. Multiply charged inorganic anion can be added in the form of corresponding salts, for example sodium salts, potassium salts and their mixtures and/or in the form of a buffer, e.g. a phosphate buffer.

The concept of "isoosmotic or isotonic" refers to a solution which can be mixed with the total body water, without affecting its components. Solutions that are isotonic with blood, such as a 0.9%solution of sodium chloride, have the same osmotic pressure as blood serum, and they do not affect erythrocyte membranes. Generally, solutions that are isotonic with blood, have an osmotic pressure of approximately 290 mOsm/kg H2 O.

The concept of "strong inorganic acid" refers to inorganic acid dissociation which in 1H. the solution is from 20 to 100%, for example, H2SO4.

The term "pharmaceutically acceptable" in the context of the present description means that the buffer or salt are acceptable from the standpoint of toxicity.

The term "diluent" refers to part of a medical drug ingredient, which does not possess pharmacological activity but which is necessary or desirable from a pharmaceutical point of view. For example, the diluent may be a liquid for dissolving intended(s) for injection drug(governmental) funds(C), for example water.

The term "solvent" refers to a liquid that contains dissolved in another substance, i.e. it dissolves, for example to the water.

The term "preservative" refers to a substance which is added to the pharmaceutical composition to prevent the growth of bacteria, such as benzylaniline or benzyl alcohol.

The term "polyol" refers to any substance that carries several hydroxyl groups, including polyhydric alcohols and carbohydrates. Polyhydric alcohols include compounds such as sorbitol, mannitol and glycerol. Carbohydrates are cyclic molecules carrying the UTB - or aldehyde group, such as sucrose or trehalose.

The term "erythropoietin" or "protein erythropoietin" refers to the protein having the in vivo biological activity, resulting in bone-marrow cells to increase production of reticulocytes and red blood cells, which are selected from the group comprising human erythropoietin and its analogues, which are described below.

The concept of "targeted erythropoietin (Peg-EPO or PEG-EPO) refers to the protein erythropoietin, covalently linked with one or three derivatives of polyethylene, as described below.

The term "device" means a device designed for a particular purpose. In the context of the present invention the goal is to implement, maintain or facilitate the introduction of liquid pharmaceutical compositions.

Description of the drawings

Figure 1: Primary structure of human EPO (165 amino acids) (SEQ ID NO:1).

Figure 2: Primary structure of human EPO (166 amino acids) (SEQ ID NO:2).

Figure 3: Effect of pH on thermal stability. Presents a plot of the phase transition temperature from pH values.

Figure 4: Influence of ionic strength on the stability. Presents a plot of the phase transition temperature on the concentration of phosphate.

Figure 5: Dependence of thermal stability on the type of buffer substance.

F is 6: data, suggesting that sulfate is also acceptable buffer/additive at low pH values (for example, when the pH of 6.2), while phosphate at pH 6,2 less effective than at pH 7.5. This suggests that sulfate provides a high level of thermal stability even at low pH values.

7: dependence of the aggregation of PEG-EPO from pH. Samples of PEG-EPO after heat stress (as described above) were analyzed using SDS page-ordinator. Proteins were stained with silver. Lane 1: standard molecular weight. Lane 2: pH 5. Band 3: pH 5, after recovery. Band 4: pH 6. Band 5: pH 6, after recovery. Band 6: pH 6.5. Band 7: pH 6.5, after recovery. Band 8: pH 7. Band 9: pH 7, after recovery. Band 10: PEG-EPO, without heat stress.

Fig: the above data that the use of acetylcysteine in a concentration of 1 mg/ml as an antioxidant prevents the formation of aggregates under conditions of heat stress. Aggregation of PEG-EPO in conditions of heat stress (80°C for 20 min): band 1: PEG-EPO at pH 7.5, without stress; lane 2: PEG-EPO at pH 7.5, in stress conditions; lane 3: PEG-EPO at pH of 6.2, in stress conditions; lane 4: PEG-EPO at pH of 6.2, under stress, after recovery; lane 5: PEG-EPO at pH 7.5+1 mg/ml N-acetylcysteine, under stress; lane 6: PEG-EPO at pH 7.5+1 mg/ml N, under stress, after recovery.

Fig.9: the Content of sialic acid (NANA) in samples of new songs, including PEG-EPO, stored for 6 months at different temperatures.

Figure 10: Analysis in mice biological activity of samples containing PEG-EPO, which was stored for 6 months at different temperatures in 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2.

11: Comparison of chromatograms obtained by the method of gel filtration, for samples containing PEG-EPO, which was stored for 6 months at different temperatures in 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2 (top to bottom: the buffer, the original product, 4°, 25°, 30°s and 40°).

More specifically, the present invention relates to a liquid pharmaceutical composition containing the protein erythropoietin, multiply charged inorganic anion in a pharmaceutically acceptable buffer suitable for maintaining the pH of the solution in the range from approximately 5.5 to approximately 7,0, and optionally one or more pharmaceutically acceptable excipients.

In a preferred embodiment, the composition is a liquid solution such as an aqueous solution. In a preferred embodiment, the above pharmaceutical to the notizie are isotonic solutions.

Anion preferably chosen from anions of strong inorganic acids, such as H2SO4N3PO4or citric acid. Thus, the preferred anions are selected from the group including sulfate, phosphate and citrate, preferred is a sulfate or phosphate, the most preferable is the sulfate. The concentration of multiply charged inorganic anion can be from 10 to 200 mmol/l, for example, sulfate anion, it can be from 10 to 200 mmol/L.

As the buffer used according to the invention to maintain the pH from about 5.5 to about 7.0 and preferably from about 5.8 to 6.7, more preferably from 6.0 to 6.5 and most preferably at the level of about 6.2, you can apply the regular buffers on the basis of organic or inorganic acids (for example, phosphate buffer, arginine/N2SO4/Na2SO4buffer or any other pharmaceutically acceptable buffer system). In a preferred embodiment, the composition comprises a phosphate buffer or arginine/N2SO4/Na2SO4buffer, preferably a phosphate buffer with a concentration of 10-50 mmol/L. Obviously, combinations of these buffer systems also fall under the scope of the present invention. The pH value can be adjusted using COO the relevant grounds, for example, NaOH, in a phosphate buffer system, and the appropriate acid, for example sulphuric acid arginine buffer system, respectively.

The compositions of the present invention may contain one or more pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients can be selected from the group comprising pharmaceutically acceptable salts, diluents and/or solvents, and/or preservatives, etc., for example, agents for regulating toychest (agents, providing isotonicity), polyols, antioxidants or nonionic surfactants. Examples of such substances are sodium chloride, calcium chloride, sorbitol, mannitol, glycerol, sucrose, trehalose, acetylcysteine, Polysorbate 20, Polysorbate 80 or pluronic F68.

In a preferred embodiment of the present invention the pharmaceutical composition may contain a polyol selected from the group comprising mannitol, sorbitol, glycerol, trehalose and sucrose, preferably mannitol. The concentration of the polyol may comprise 1-10% (wt./vol.).

Examples of antioxidants are cysteine, methionine, acetylcysteine or ascorbic acid, preferably methionine. Antioxidants, as a rule, can be added in a concentration of from 0.01 to 0.5% (wt./vol.), or, for example, in the case of methionine at a concentration of 1-20 the M.

The above composition optionally can include providing isotonicity agent in an amount of from about 0.01 to about 0.9 wt.%. Such compounds are known in the art; examples of such agents are sodium chloride or sodium sulfate. In a preferred embodiment of the present invention compositions are isotonic solutions. The above compositions can also contain nonionic detergent Polysorbate 20, Polysorbate 80 or pluronic F68, preferably pluronic F68, for example, in an amount up to 1% (wt./vol.), more preferably up to 0.1% (wt./vol.), for example, from 0.001 to 0.01% (wt./vol.).

The above composition may also contain other salts, for example, up to 1 mmole/l CaCl2.

The present invention relates primarily to obtain pharmaceutical compositions containing as having a pharmaceutical active ingredient erythropoietin. The term "erythropoietin" or "protein erythropoietin" or "EPO" means: these relate primarily to the glycoprotein, for example, human erythropoietin, for example, having the amino acid sequence represented in SEQ ID NO:1 or SEQ ID NO:2, or amino acid sequence, almost homologous to these sequences, biologists the definition of the properties which cause stimulation of the production of red blood cells and stimulate the division and differentiation of commiteeman erythroid precursors in the bone marrow. In the context of the present description these concepts include proteins that are intentionally modified, for example, using siteprovides mutagenesis, or as a result of random mutations. These concepts also include analogs having from 1 to 6 additional sites of glycosylation analogs having at least one additional amino acid at the carboxyl end of the peptide, and the additional amino acid includes at least one glycosylation site, and analogs having amino acid sequence, which includes a rearrangement of at least one glycosylation site. These concepts include both naturally occurring and obtained by recombinant human erythropoietin.

As described in detail below, methods of obtaining and purification of EPO is well known in this field. Erythropoietin is a naturally occurring or recombinant protein, preferably human, which can be obtained from any available source, for example, from the tissues, by the synthesis of the protein from cell culture using naturally occurring or recombinant cells. This term includes any protein having the activity of erythropoietin, for example, from the group of Malinov or other education is ω-modified proteins. Recombinant EPO can be obtained by expression in the cell lines Cho, KSS (kidney cells baby hamster) or HeLa (culture of tumor cells Helen lake), recombinant DNA, or by endogenous gene activation. Methods the expression of proteins, including endogenous activation of the gene are well known in this field and are described, for example, in patents US 5733761, 5641670 and 5733746 and in published international patent applications WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91/09955, the content of each of these documents is hereby incorporated into this description by reference. The preferred types of EPO to obtain products containing glycoprotein erythropoietin, are types of human EPO. More preferably the kinds of EPO are human EPO having the amino acid sequence represented in SEQ ID NO:1 or SEQ ID NO:2, more preferably the amino acid sequence represented in SEQ ID NO:1.

In addition, erythropoietin may be a similar glycoprotein having from 1 to 6 additional sites of glycosylation. Glycosylation of proteins carrying one or more oligosaccharide groups, occurs in certain provisions of the polypeptide on the frame and has a strong influence on the physical properties of the protein, such as protein stability, secretion, nutrilett is Chou localization and biological activity. There are two types of glycosylation. O-linked oligosaccharides are associated with the remnants of serine or threonine, and N-linked oligosaccharides linked to asparagine residues. One of the types of oligosaccharides found in N-linked and O-linked the oligosaccharide is N-acetylneuraminic acid (sialic acid), which belongs to the family of amino sugars, carrying 9 or more carbon atoms. Sialic acid, as a rule, represents a limit balance as on N-linked and O-linked-oligosaccharides and because it carries a negative charge, it gives the acid glycoprotein properties. Human erythropoietin with 165 amino acids, contains three N-linked and one O-linked oligosaccharide chain, which accounted for approximately 40% of the total molecular weight of the glycoprotein. Glycosylation N-linked oligosaccharides occurs at asparagine residues at positions 24, 38 and 83, and glycosylation O-linked oligosaccharides occurs on serine residue at position 126. Terminal sialic acid residues modify the oligosaccharide chain. Removal using enzymes all sialic acid residues of the glycosylated erythropoietin leads to loss of activity in vivo, but does not lead to loss of activity in vitro, as sililirovanie erythropoietin prevents its binding and will follow the him clearance hepatic binding protein.

The term "erythropoietin", used in relation to the pharmaceutical compositions of the present invention includes analogs of human erythropoietin having one or more substitutions in the amino acid sequence of human erythropoietin, which leads to an increase in the number of binding sites with sialic acid. Such glycoprotein analogs can be created by siteprovides mutagenesis, leading to additions, deletions, or substitutions of amino acid residues, causing an increase in the number or the modification of sites suitable for glycosylation. Glycoprotein analogues having a content of sialic acid, more than content, characteristic of human erythropoietin, create by introducing sites that do not disrupt the secondary or tertiary conformation that determine their biological activity. The glycoproteins of the present invention also include analogs having elevated levels of attachment of carbohydrates in the site of glycosylation, which, as a rule, are replacing one or more amino acids near the N-linked or O-linked site. The glycoproteins of the present invention also include analogs having one or more amino acids, extending from the C-end of erythropoietin and having at least about whom in additional carbohydrate website. Proteins erythropoetin included in the compositions of the present invention also include analogs having amino acid sequence, which is characterized by the rearrangement of at least one glycosylation site. Such regrouping site of glycosylation represents a deletion of one or more glycosylation sites in human erythropoietin and adding one or more glycosylation sites not found in natural conditions. Increasing the number of carbohydrate chains on erythropoietin and, consequently, the number of residues of sialic acid per molecule of erythropoietin can give valuable properties, such as higher solubility, increased resistance to proteolysis, reduced immunogenicity, increasing the half-life in serum and higher biological activity. Analogs of erythropoietin, with additional sites of glycosylation are described in more detail in the application for the European patent 640619 in the name of Elliot, published March 1, 1995

In a preferred embodiment, the pharmaceutical composition of the present invention contains proteins erythropoetin having the amino acid sequence which contains at least one additional glycosylation site, such as erythropoetin, is within (but not limited to) the sequence of human erythropoietin, including modification selected from among the following modifications:

Asn30Thr32,

Asn51Thr53,

Asn57Thr59,

Asn69,

Asn69Thr71,

Ser68Asn69Thr71,

Val87Asn88Thr90,

Ser87Asn88Thr90,

Ser87Asn88Gly89Thr90,

Ser87Asn88Thr90Thr92,

Ser87Asn88Thr90Ala162,

Asn69Thr71Ser87Asn88Thr90,

Asn30Thr32Val87Asn88Thr90,

Asn89Ile90Thr91,

Ser87Asn89Ile90Thr91,

Asn136Thr138,

Asn138Thr140.

Thr125and

Pro124Thr125.

Conventions used in this description to indicate modification of the amino acid sequence, means that the amino acid(s) in position(s) sequence of the corresponding unmodified protein (e.g., SEQ ID NO:1 or SEQ ID NO:2 Nero)listed the top(and) index(AMI) is replaced by the amino acid(s)found(s) directly before the corresponding upper index.

The protein erythropoietin may be also similar, with at least one additional amino acid at the carboxyl end of the peptide, and the additional amino acid which engages at least one glycosylation site. Additional amino acid sequence may include a peptide fragment derived from the carboxyl end related to human chorionic gonadotropin. Preferably, the glycoprotein is an analogue selected from the group comprising (a) human erythropoietin having the amino acid sequence SerSerSerSerLysAlaProProProSerLeuproserproserargleuproglyproseraspthrproileleuprogln (SEQ ID NO:3), extending from the carboxyl end; (b) analogue specified in section (a), optionally including Ser87Asn88Thr90EPO; and (C) similar specified in section (a), optionally including Asn30Thr32Val87Asn88Thr90EPO.

The protein erythropoietin may also be analogous to having the amino acid sequence, which includes a rearrangement of at least one glycosylation site. The rearrangement can be a deletion of any of the N-linked carbohydrate sites in human erythropoietin and the addition of N-linked carbohydrate site at position 88 the amino acid sequence of human erythropoietin. Preferably, the glycoprotein is an analogue selected from the group comprising Gln24Ser87Asn88Thr90EPO; Gln38Ser87Asn88Thr90EPO and Gln83Ser87Asn88Thr90EPO.

In more site is Cetelem variation in the quality of the protein erythropoietin, included in the above-described pharmaceutical compositions of the present invention may be applied paglierani derivatives. Paglierani derivatives of erythropoietin and containing compositions known in this field and are described, for example, in EP-A-539167, EP-A-605963, WO 93/25212, WO 94/20069, WO 95/11924, 556 patent US. EP-A-584876, WO 92/16555, WO 94/28024, WO 97/04796, patents US 5359030 and 5681811, patent US 4179337, Japanese patent WO 98/32466, patent US 5324650. The preferred option species paglinawan erythropoietin are described below derived.

In accordance with the foregoing the present invention also relates to conjugates of erythropoietin, which contains the above-described protein erythropoietin having at least one free amino group and having the biological activity in vivo, leading to increased production of reticulocytes and red blood cells by the bone marrow cells, and which are selected from the group comprising human erythropoietin and its analogues having the sequence of human erythropoietin modified by the addition of 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; where erythropoietin covalently linked to "n" poly(etilenglikolevye) groups of the formula-CO-(CH2)x-(OCH2CH2)m-OR, in which the radical (i.e. the ka is bonyl) of each poly(ethylene)pikolinos group forms an amide bond with one of these amino groups; R denotes (ness.)alkyl; x is 2 or 3; m is a number from about 450 to about 900; n is 1-3; and n and m are chosen so that the molecular weight of the conjugate minus the molecular weight of the glycoprotein erythropoietin ranged from 20 to 100 kDa. In this proposed invention is also pharmaceutical compositions containing the above-described conjugates, in which the content of conjugates in which n is 1, is at least 90%, preferably at least 92%, more preferably 96% of the total number of all conjugates contained in the composition.

More specifically, the above conjugates can be represented by the formula (I)

where P stands above the rest of the protein erythropoietin (i.e. not containing the amino group or amino groups which form an amide bond with the carbonyl represented by the formula (I)possess biological activity in vivo, leading to increased production of reticulocytes and red blood cells by the bone marrow cells; and where R denotes the (ness.)alkyl; x is 2 or 3; m is a number from about 450 to about 900; n is 1-3; and n and m are chosen so that the molecular weight of the conjugate minus the molecular weight of the glycoprotein erythropoietin ranged from 20 to 100 kDa.

In to the texte of the present description, the term "(ness.)alkyl" oboznachat alkyl group with straight or branched chain, bearing 1-6 carbon atoms. Examples (ness.)alkyl groups are methyl, ethyl and isopropyl. According to the present invention R can be any (ness.)alkyl. Preferred are conjugates in which R represents methyl.

The symbol "m" denotes the number ethylenoxide links (och2CH2in poly(ethyleneoxide) group. One ethylenoxide subunit of PEG (polyethylene glycol) has a molecular mass of approximately 44 Yes. Thus, the molecular weight of the conjugate (excluding molecular weight of EPO) depends on the number of "m". In the conjugates of the present invention "m" is a number from about 450 to about 900 (which corresponds to a molecular weight of from about 20 to about 40 kDa), preferably from about 650 to about 750 (which corresponds to a molecular mass of approximately 30 kDa). The number m is chosen so that the resulting conjugate according to the present invention had a physiological activity comparable to the activity of unmodified EPO, with its activity may be equal, exceed or be part of the activity of unmodified EPO. The term "molecular weight is approximately" the specified value means that its value is close enough to decree the specific value, which is determined using conventional analytical methods. The number "m" is chosen so that the molecular weight of each poly(etilenglikole) groups covalently linked to the glycoprotein erythropoietin, ranged from approximately 20 to approximately 40 kDa, preferably of about 30 kDa.

In the conjugates of the present invention, the number "n" indicates the number of poly(etilenglikolevykh) groups covalently linked to free amino groups (including ε-amino group of the amino acids lysine and/or N-terminal amino group) of the protein erythropoietin using amide(s) connection(s). The conjugates of the present invention can have one, two or three PEG groups per molecule of EPO. "n" is an integer from 1 to 3, preferably n is 1 or 2, more preferably n is 1. Preferred conjugates as described above conjugates are compounds where x is 2, m denotes a number from 650 to 750, n is 1 and R is methyl.

The compounds of formula (I) can be obtained from known polymers:

where R and m have the above values, by condensation of compounds of formula II with the glycoprotein erythropoietin. The compounds of formula (II)in which x is 3, represent Succinimidyl esters of alpha(ness.)alkoxyl(ethylene glycol) and the oil is Noah acid ((ness.)alkoxy-PEG-QMS). The compounds of formula (II)in which x is equal to 2, are Succinimidyl esters of alpha(ness.)alkoxyl(ethylene glycol) and propionic acid ((ness.)alkoxy-PEG-SEC). To obtain amide, you can use any of the usual methods for the interactions of the activated ester with the amine. In the reaction described above is specified as an example Succinimidyl ether is a leaving group, allowing the amide formation. Application Succinimidyl esters, such as the compounds of formula II to obtain conjugates with proteins described in the patent US 5672662, issued September 30, 1997 (in the name of Harris, and others).

Human EPO contains nine free amino groups, i.e. the N-terminal amino group plus ε-amino group of 8 lysine residues. It was found that when PageLime reagent combined with QMS-derived formula II, when the pH value of 7.5, the ratio of protein: PEG = 1:3, and the reaction temperature 20-25°To form a mixture containing mono-, di -, and trace amounts trippelironi products. If PageLime reagent represented SPK-derived formula II, in similar conditions, except that the ratio of protein: PEG was 1:2, received mostly monopegylated products. Targeted EPO can be entered in the form of a mixture or in the form of various who's paglierani species extracted using cation exchange chromatography. By manipulating the reaction conditions (e.g., ratio of reagents, pH, temperature, protein concentration, duration of response, and so on) you can change the relative number of different paglierani products.

The above-described pharmaceutical compositions can also contain the above protein erythropoietin having at least one free amino group and having the biological activity in vivo, leading to increased production of reticulocytes and red blood cells by the bone marrow cells, and which are selected from the group comprising human erythropoietin and its analogues having the primary structure of human erythropoietin modified by the addition of 1 to 6 glycosylation sites; where the glycoprotein covalently associated with one-three (ness.)alkoxyl(etilenglikolevye) groups, each poly(etilenglikolya) group covalently linked to the glycoprotein using the linker of the formula-C(O)-X-S-Y - fragment(O) of the linker, resulting in an amide bond with one of the amine groups, X represents -(CH2)k- or-CH2(O-CH2-CH2)k-, k is 1 to 10, Y represents

or

the average molecular weight of each poly(etilenglikole) fragment is from about 20 to about 40 kDa, and the molecular mass of the conjugate is from about 51 to about 175 kDa. These types of erythropoietin can be represented by formula (III)

where R can be any (ness.)alkyl, i.e. alkyl group with straight or branched chain, carrying one to six carbon atoms, such as methyl, ethyl, isopropyl, etc. Preferably alkyl represents methyl. X can be a -(CH2)k-or-CH2(O-CH2-CH2)k-where k has a value from 1 to about 10. Preferably k has a value from 1 to about 4, more preferably k is 1 or 2. Most preferably X represents -(CH2).

In the formula 1 Y denotes

or

preferably

or

more preferably

In the formula (III), the number m is chosen so that the resulting conjugate of formula (III) had physiological activity comparable to the activity of named fitiavana EPO, however, his activity may be equal, exceed or be part of the activity of unmodified EPO. m denotes the number ethyleneoxide units per unit of PEG. One subunit of the PEG, i.e. -(och2CH2)-has a molecular weight of approximately 44 Yes. Thus, the molecular weight of the conjugate (excluding molecular weight of EPO) depends on the number m. The term "molecular weight is approximately" the specified value means that its value is close enough to the specified value that is determined using conventional analytical methods, m denotes an integer from about 450 to about 900 (which corresponds to a molecular weight from 20 to 40 kDa), preferably m is from 550 to about 800 (which corresponds to a molecular weight of from about 24 to 35 kDa), and most preferably m is from about 650 to about 700 (which corresponds to a molecular weight of from about 29 to about 31 kDa).

In the formula (III), n represents the number ε-amino group of the amino acid lysine in the protein erythropoietin, covalently linked with unit PEG through amide linkages. The conjugate of the present invention can have one, two or three edges of the PEG molecule of EPO. n denotes an integer from 1 to 3, preferably n is 1 or 2, Nai is more preferably n is 1.

Preferred proteins erythropoetin formula (III) are described by the formulas:

and

The most preferred products containing glycoprotein erythropoietin represented by the formula:

where n in the above formula denotes an integer from 1 to 3; m represents an integer from 450 to 900; R is (ness.)alkyl; X represents -(CH2)k- or-CH2(O-CH2-CH2)kand R denotes the residue of the glycoprotein erythropoietin without the amino group or groups, forming an amide bond with X.

Other preferred products containing glycoprotein erythropoietin represented by the formula:

and

Preferred products containing glycoprotein erythropoietin represented by the formula:

Such proteins erythropoetin can be obtained by

(a) covalent interaction ε-amino group of the amino acid lysine protein erythropoietin represented by the formula P-[NH2]nwith a bifunctional reagent represented by the formula Z-CO-X-S-Q, to obtain an intermediate product having an amide bond represented by the formula:

where P convoy is achet protein erythropoietin, not containing aminogroup, forming an amide bond; n denotes an integer from 1 to 3; Z represents reactive group, for example, NHS-ester carboxylic acid; X represents -(CH2)k- or-CH2(O-CH2-CH2)k-where k denotes a number from 1 to about 10; and Q represents a protective group of type alkanoyl, for example, acetyl;

(b) covalent interaction of the intermediate product having an amide bond, obtained in stage (a)with activated polietilenglikoli derivative represented by the formula W-[OCH2CH2]m-OR, with the receipt containing the glycoprotein erythropoietin product represented by the formula:

where W denotes a sulfhydryl reactive form Y; m denotes an integer from about 450 to about 900; R denotes (ness.)alkyl; and Y has the above values.

According to this variant bifunctional reagent preferably represents N-Succinimidyl-S-acetylthiophene or N-Succinimidyl-S-acetylthiourea, Z preferably denotes N-hydroxysuccinimide and activated poliatilenglikole derived W-[OCH2CH2]m-OR preferably chosen from the group consisting of todatetimeoffset-PEG, methoxy-PEG-vinylsulfonic and methoxy-PEG-maleimide.

More specifically, PR is teeny erythropoetin formula (III) can be obtained by covalent binding tylnej groups with EPO ("activation") and combining the resulting activated EPO with poly(etilenglikolem) (PEG) derivative. The first stage of obtaining paglinawan EPO of the present invention provides covalent binding tylnej groups with NH2groups EPO. Such activation of the EPO carried out using bifunctional reagents, which are protected Tilney group and an additional reactive group, selected from a range that includes possessing activity esters (for example, Succinimidyl ester, anhydrides, esters, sulfonic acid halides of carboxylic acids and sulfonic acids, respectively. Tilney group protects with famous in this area, for example, acetyl group. Such bifunctional reagents have the ability to interact with ξ-amino groups of the amino acids lysine through the formation of amide linkages. The first stage of the reaction is presented below:

EPO, n and X have the above values, Z represents a reactive group, known in this area, for example, N-hydroxysuccinimidyl (NHS) Deputy formula

In a preferred embodiment, activation ε-amino group of lysine is carried out by interaction with bifunctional reagents containing Succinimidyl fragment. Bifunctional reagents can carry a variety of t is s spacers, for example, -(CH2)k- or-CH2-(O-CH2-CH2-)kfragments, where k has a value from 1 to about 10, preferably from 1 to about 4, more preferably 1 or 2, and most preferably 1. Examples of such reagents are N-Succinimidyl-S-acetylthiophene (SATP) and N-Succinimidyl-S-acetylthiourea (SATA)

where k has the above values.

Methods for obtaining a bifunctional reagents known in the field. Predecessors NHS-esters of 2-(acetylthio)(ethoxy)kacetic acid is described in DE-3924705 and derivatization method of obtaining compounds containing acetylthiourea described in J. March, Advanced Organic Chemistry, McGraw-Hill, 375-376, 1977. SATA is available (the company Molecular Probes, Eugene, Oregon, USA and firm Pierce, Rockford, Illinois).

In the molecule of EPO, you can enter the required number tylnej groups by adjusting the parameters of the reaction, i.e. the concentration of protein (EPO) and the ratio of protein/bifunctional reagent. Preferably EPO is activated by covalent binding 1-5 tylnej groups from one molecule of EPO, more preferably from 1.5 to 3 tylnej groups from one molecule of EPO. These ranges characterize the statistical distribution of the thiol which groups in the population of EPO protein.

The reaction is carried out, for example, in an aqueous buffer solution, pH 6.5 to 8.0, for example, in 10 mm potassium phosphate, 50 mm NaCl, pH to 7.3. Bifunctional reagent can be added in DMSO. After the reaction for approximately 30 min the reaction is stopped by adding lysine. An excess of a bifunctional reagent can be removed by methods known in this field, for example, by dialysis or filtration on columns. The average number tylnej groups attached to the EPO, it is possible to determine photometric methods described, for example, D.R.Grasetti and J.F.Murray in J. Appl. Biochem. Biotechnol. 119, 41-49 (1967).

After carrying out the reaction described above provide covalent binding of activated poly(etilenglikole) (PEG) derivative. Suitable PEG-derivatives are activated molecules of PEG with an average molecular weight of from about 20 to about 40 kDa, more preferably from about 24 to about 35 kDa, and most preferably approximately 30 kDa.

Activated PEG derivatives known in this field, for example, Mogridge etc., J. Bioconj. Chem. 7, str and beyond (1996) described PEG-vinylsulfonic. To obtain compounds of formula 1 can be applied types of PEG with linear and branched chain. Examples of reactive PEG reagents are todatetimeoffset-PEG and methoxy-PEG-vinyls Levon:

or

The use of such activated iodine substances known in this field and are described, for example, G.T.Hermanson in Bioconjugate Techniques, Academic Press, San Diego, str-148 (1996).

Most preferably different tweed PEG activate using maleimide using (alkoxy-PEG-maleimide), such as methoxy-PEG-maleimide (MM 30000; firm Shearwater Polymers, Inc.). Alkoxy-PEG-maleimide has the following structure:

or

where R and m have the above values, preferably

The reaction mix with alkoxy-PEG-maleimide occurs after removal of the in situ tylenol protective groups in aqueous buffer solution, for example, containing 10 mm potassium phosphate, 50 mm NaCl, 2 mm add, pH of 6.2. The removal of the protective group can be performed, for example, by treatment with hydroxylamine in DMSO at 25°With a pH of 6.2, for about 90 minutes For modification of PEG molar ratio of activated EPO/alkoxy-PEG-maleimide should be from about 1:3 to about 1:6, preferably 1:4. The reaction can be stopped by adding cysteine and interaction remaining tylnej (-SH) groups with N-methylmaleimide or other acceptable compounds that can form disulfide the e connection. Due to the interaction of any remaining active tylnej group with a protective group, such as N-methylmaleimide, or other appropriate protective group, glycoproteins EPO in the conjugates of the present invention may contain such protective groups. Generally, the described process results in a mixture of molecules having different number tylnej groups that are protected under different number of protective groups, which depends on the number of activated tylnej groups that are not conjugated with PEG-maleimide.

While N-methylmaleimide when it is used to block the remaining tirinya group paglinawan protein forms a covalent bond of the same type, disulfide compounds should lead in the intramolecular exchange reaction sulfide/disulfide binding by a disulfide bridge blocking reagent. Preferred blocking agents for this type of reaction block are oxidized glutathione (GSSG), cysteine and tsistamin. While cysteine does not introduce additional net charge in pegylated protein, the use of blocking reagents GSSG or applied leads to more negative or positive charge.

Further purification of the compounds of the formula (III), including split the mono-, di - and trippelironi types EPO, can be accomplished by methods well known in this field, for example, chromatography on columns.

A composition including paglierani derived erythropoietin, preferably containing at least 90% of Tonopah-conjugates, i.e. in which n is 1, can be obtained according to the method described in example 5. As a rule, preferred are nanopeg-glycoprotein conjugates of erythropoietin, as they are characterized by a higher activity than dipag-conjugates. The percentage of Tonopah-conjugates, and the ratio of mono - and djpeg-types can be adjusted by combining the fractions, or located in a wider range around the peak of the elution in order to reduce the percentage of Tonopah, or in a more narrow range with the aim of increasing the percentage of Tonopah in the composition. A satisfactory ratio of output and activity is achieved if present, approximately 90% of Tonopah-conjugates. In certain cases it may be desirable to apply the composition in which, for example, at least 92% or at least 96% of the conjugates belong to Tonopah-mean (n = 1). According to one of embodiments of the present invention, the percentage of conjugates in which n is , ranges from 90% to 96%.

The compositions of the present invention may contain from 10 to 10000 μg of the above protein erythropoietin in ml. Preferably the compositions contain from 10 to 1000 μg, for example, 10, 50, 100, 400, 800 or 2500 m to g/ml.

In addition, the compositions of the present invention may contain from 10 to 10,000 μg protein erythropoietin per ml, 10-200 mmol/l sulfate, 10 to 50 mmol/l phosphate, pH 6.0 to 6.5. This composition may also contain up to 20 mm methionine, 1-5% (wt./about.) polyol, up to 0.1% (wt./about.) pluronic F68 and optional up to 1 mm CaCl2. An example of such a composition is a composition containing 10-10000 mg of protein erythropoietin per ml, 40 mmol/l of sulfate, 10 mmol/l phosphate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2.

According to another variant implementation of the present invention the composition may contain 10-10000 mg of protein erythropoietin per ml, 10-100 moles/l NaCl, 10 to 50 mmol/l phosphate, pH 6,0-7,0, optional 1-5% (wt./about.) polyol. In addition, the composition may contain up to 20 mm methionine, 0.1% (wt./about.) pluronic F68 and optional 7.5 mmol/l CaCl2. According to one of embodiments, such a composition may contain 10-10000 mg of protein erythropoietin per ml, 100 mol/l NaCl, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68 and 10 mmol/l phosphate, pH 7.0.

The present invention relative to the tsya to the above composition, containing 10-10000 mg of protein erythropoietin per ml, 10-50 mmol/l arginine, pH 6-6,5, 10-100 mmol/l of sodium sulfate. In addition, the composition may contain up to 20 mm methionine, 0.1% (wt./about.) pluronic F68, optional up to 1 mmol/l CaCl2and optional 1-5% (wt./about.) polyol. According to one of the embodiments mentioned composition may contain 10-10000 mg of protein erythropoietin per ml, 40 mmol/l arginine, pH 6,2, 30 mmol/l of sodium sulfate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, optional 1 mmol/l CaCl2.

According to a preferred variant implementation of the present invention the compositions contain 10-10000 mg/ml erythropoietin, preferably 25-2500 µg/ml erythropoietin, and

a) 10 mm sodium phosphate/potassium, 100 mm NaCl, pH 7.0 or

b) 10 mm sodium phosphate, 120 mm sodium sulfate, pH of 6.2 or

in) 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2 or

g) 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2 or

d) 40 mm arginine, 30 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2 or

e) 40 mm arginine, 30 mm sodium sulfate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2.

According to a preferred variant implementation compositions contain 50, 100, 400, 800 or 2500 µg/ml of protein erythroped is on. The most preferred compositions contain either 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2 or 40 mm arginine, 30 mm sodium sulfate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2.

The compositions of the present invention can be in the form of a spray dried powder.

In addition, the invention relates to compositions, representing a lyophilized or spray dried powder having the above composition. The composition can be restored by receiving a liquid solution by adding a solvent, such as water, or they can be used directly, for example, using the device for inhalation or device for transdermal administration.

The invention relates also to a method for producing the above-described composition, providing a mixture of protein erythropoietin with a solution containing carries many negative charges on the anion, and optionally one or more of the above pharmaceutically acceptable excipients.

In addition, the present invention relates also to the use of the above compositions for the preparation of drugs that can be used for the treatment and prevention of diseases associated with anemia in patients with chronic renal under what tochnostu (CRF), AIDS patients and/or to treat cancer patients undergoing chemotherapy. In addition, the invention relates to a method for the treatment and prevention of diseases involving anemia in patients with chronic renal failure (CRF), AIDS patients and cancer patients undergoing chemotherapy to which the stages of the introduction of the patient described above composition.

The following variant of implementation of the present invention relates to a device for local and systemic continuous administration of medicinal products containing the above composition. The device may be any type of implant that provides controlled release of erythropoietin, which is included in the above-described composition, such as micro - or nanoparticles based on the polymer. The above composition can also be in pre-filled syringe or any other device for administration, such as, for example, without needle device for injection or device for inhalation.

The composition according to the invention can be in the form of standard dosage forms intended for injection or intravenous administration. On the other hand, the composition according to the invention can be stored in liquid or solid form and then divide by the standard dosage forms, the purpose is authorized for injection or intravenous administration. Therefore, the liquid composition of the present invention may be present in an amount of at least 0.3 ml to ensure that it can be used for introduction as a standard dosage forms. On the other hand, the amount of the claimed compositions can reach 60 l, if it is stored before distribution in Packed dosed form. Taking into account its increased stability, the composition according to the invention can be stored in large containers in order to later be placed in a package suitable for distribution to doctors, patients and hospitals.

Injectable solution according to the present invention can be entered using a conventional devices for injection, such as syringes, allowing you to enter from 0.3 to 20 ml of the composition in the form of a standard dose. On the other hand, the composition according to the invention can be administered by injection, using capsules that contain the composition either in the form of a freeze-dried or spray dried powder, which restores before injection usual method. In addition, due to their stability, the compositions of the present invention can be packaged in standard dosage forms, such as bubbles, containing from about 0.3 to about 10 ml of the indicated composition. In addition, the compositions of the present invention can be administered intravenously with IP is the use of containers for intravenous administration. Such containers contain from about 20 to about 500 ml, depending on the period of time during which the solution is required to enter the patient. According to the invention a liquid solution according to the invention can be stored in a designated storage containers, from which it can then be packaged in a small package for dosage forms intended for distribution to doctors, patients and clinics. Due to the stability of the compositions according to the invention, these compositions can be stored in such containers for extended periods of time prior to the introduction.

Receiving erythropoietin as an ingredient described above compositions or as the source of the product to obtain the above-described derivatives of erythropoietin are described in detail, for example, in patents US 5547933 and 5621080, EP-0148605, S.L.Huang. Proc. Natl. Acad. Sci. USA 2708-2712 (1984), in EP-0205564, EP-0209539 and EP-0411678, and ..Lai etc., J. Biol. Chem. 261, 3116-3121 (1986), .Sasaki etc., J. Biol. Chem. 262, 12059-12076 (1987). For therapeutic purposes erythropoietin can be obtained recombinant methods (see EP-0148605. EP-0209539 and J.C.Egrie, T.W.Strickland, J.Lane, etc., Immunobiol. 72: 213-224 (1986)).

Methods expression and receiving erythropoietin in serum-free medium is described, for example, in WO 96/35718 on the name Burg, published on 14 November 1996, and in European patent No. 513738 on the name Koch, published on 12 June 1992. SIP the mo methods, described in the above publications, it is known that it is possible to carry out the fermentation in serum-free medium of recombinant cells SNO, which contain the gene for EPO. Such methods are described, for example, in EP-A 0513738, EP-A 0267678 and in General form in .Kawamoto and others, Analytical Biochem. 130, 445-453 (1983), in EP-A 0248656, J.Kowar and F.Franek, Methods in Enzymology 421, 277-292 (1986), .Bavister, Expcology 271, 45-51 (1981), in EP-A 0481791, EP-A 0307247, EP-A 0343635, WO 88/00967.

In EP-A 0267678 describes the purification of EPO obtained in serum-free culture after dialysis, which is carried out using ion-exchange chromatography on S-sepharose, preparative GHUR with reversed phase C8-column and gel filtration. In this case, the stage of gel filtration can be replaced by ion exchange chromatography on S-sepharose with great skorosti flow. Suggested before ion-exchange chromatography to perform chromatography using a column filled with Blue Trisacryl.

Method of purification of recombinant EPO described in I. Nobuo and others, J. Biochem. 107, 352-359 (1990). However, according to this method, before the stages of purification of EPO treated with a solution of Tween®20, phenylmethylsulfonyl, ethylmaleimide, pepstatin And copper sulfate and examinados acid. In several publications, including in the application WO 96/35718 on the name Burg, published on November 14, 1996, describes a method of obtaining erythropoietin by fermentation in bessboro is offered by the environment (EPOsf).

The specific activity of EPO or EPO conjugates of the present invention can determine various well-known in the field methods. Biological activity of purified proteins EPO of the present invention is that the introduction by injection of EPO protein sick people leads to the fact that the cells in their bone marrow increase production of reticulocytes and red blood cells compared with the patient groups, which were not subjected to injection, or with control groups of patients. The biological activity of proteins EPO or parts thereof, obtained and purified in accordance with the present invention, it is possible to test the methods described in Annable, etc., Bull. Wid. Hith. Org., 47: 99-112 (1972) and Pharm. Europa Spec. Issue Erythropoietin BRP Bio 1997(2). Another biological method of analysis designed to assess the activity of the protein of EPO using normocytic mice described in example 6.

The invention is explained in more detail using the following examples which serve to illustrate the invention, but not intended to limit its scope.

Examples

Example 1: Fermentation and purification of human EPO

a) Obtaining inoculum and fermentation

From the gas phase of the storage container cooled with liquid nitrogen, take one vial containing cells of the working of the Bank originating from producing EPO if the AI cells Cho (for this purpose you can use the cell line was ATSS CRL8695, described in EP 411678 (Genetics Institute)). Cell transfer in a rotating glass flask and cultured in bicarbonate buffered medium in an incubator in humidified atmosphere containing CO2. Typical serum-free environment that you can use to obtain the inoculum and fermentation, as described in the application for the European patent 513738 on the name Koch, published June 12, 1992, or in the application WO 96/35718 on the name Burg, published on 14 November 1996, for example, they include DMEM/F12 (for example, supplied by the firm JRH Biosciences/Hazleton Biologics, Denver, USA, order no 57-736), and optionally contain sodium bicarbonate, L-glutamine, D-glucose, recombinant insulin, sodium Selenite, diaminobutane, hydrocortisone, iron sulfate(II), asparagine, aspartame acid, serine and stabilizer for mammalian cells, such as polyvinyl alcohol, methylcellulose, polydextran, poly(ethylene glycol), pluronic F68, extender plasma polygeline (HEMACCEL®) or polyvinylpyrrolidone (WO 96/35718).

Culture check under the microscope regarding the absence of contaminating microorganisms and determine cell density. These tests are conducted at each stage of division.

After an initial stage of growth of the cell culture was diluted with fresh medium to the initial density of the cells and begin a new cycle of growth. This procedure is repeated until p is p, as the volume of culture will not be approximately 2 l of each rotating glass flask. After approximately 12 doublings, you can get 1-5 liters of this culture, which is then used as inoculum for the fermenter with a volume of 10 l was used for more party inoculum.

After 3-5 days of culture, grown in a fermenter with a volume of 10 l, can be used as inoculum for the fermenter with a volume of 100 l was used for more party inoculum.

After 3-5 days of culture, grown in a fermenter with a volume of 100 l, can be used as inoculum for the fermenter with a volume of 1000 l, the employee to obtain a product.

b) the Collection and separation of cells

Use a periodic process with water, this means that when reaches the desired density of cells, then collect approximately 80% culture. The rest of the culture is supplemented with fresh medium and cultured until the next collection. One cycle of receipt of the products includes a maximum of 10 consecutive charges: 9 partial charges and 1 full collection at the end of fermentation. The collection carried out every 3-4 days.

Certain the collected amount is transferred into a cooled vessel. Cells are separated by centrifugation or filtration and discarded. Containing EPO supernatant obtained at the stage of centrifugation, immediately subjected to filtration and sobi is up in the second cooling vessel. In the process of cleaning every assembled party is treated separately.

A typical cleaning process protein EPO described in the application WO 96/35718 on the name Burg, published 14 November 1996. The cleaning process is as follows.

(a) Chromatography using blue sepharose (Blue Sepharose)

Blue Sepharose, Pharmacia) consists of granules sepharose, with a surface which is covalently bonded dye Cibacron blue. Because EPO is more strongly associated with Blue Sepharose than most non-protein contaminants, some protein contaminants and PVA, the contents of EPO may be raised at this stage. Elution filled Blue Sepharose column exercise, increasing the salt concentration and pH value.

The column is filled 80-100 l Blue Sepharose, regenerate using NaOH and balance balanced buffer (sodium chloride/calcium and sodium acetate). Load obtained from the fermenter acidified and filtered supernatant. After loading the column was washed first buffer, similar to balancing a buffer that contains a higher concentration of sodium chloride, and then Tris-buffer. Product elute Tris-buffer and collected as a single fraction in accordance with the basic elution profile.

b) Chromatography using Butyl Toyopearl

Butyl Toyopearl 650 (firm Toso Haas) is a full color the COP, which is based on polystyrene, which are covalently bound aliphatic butylene fragments. Because EPO is more strongly associated with this gel than most pollutants and PVA, it can be eluted using containing isopropanol buffer.

The column is filled 30-40 l of Butyl Toyopearl 650 S, regenerate using NaOH, washed with Tris-buffer and balance with containing isopropanol Tris-buffer.

The concentration of isopropanol in the eluate obtained by chromatography using Blue Sepharose, lead to the concentration of isopropanol in the equilibrating buffer and loaded into the column. Then the column was washed with equilibrating buffer with high concentration of isopropanol. Product elute buffer for elution (Tris-buffer with high concentration of isopropanol and collected as a single fraction in accordance with the basic elution profile.

C) Chromatography using hydroxyapatite ultragas (Hydroxyapatite Ultrogel)

Hydroxyapatite Ultrogel (firm Biosepra) consists of hydroxyapatite, included in the agarose matrix to improve mechanical properties. EPO has a low affinity for hydroxyapatite and could therefore buyouts lower concentrations of phosphate than protein contaminants.

The column is filled 30-40 l Hydroxyapatite Ultrogel and regeneris the Ute buffer, containing potassium phosphate/calcium chloride and NaOH, and then Tris-buffer. Then her balance Tris-buffer with low content of isopropanol and sodium chloride.

In column load the eluate obtained by chromatography using Butyl Toyopearl. Then the column was washed with equilibrating buffer and Tris buffer without isopropanol and sodium chloride. Product elute Tris-buffer with a low content of potassium phosphate and collected as a single fraction in accordance with the basic elution profile.

g) GHUR with reversed-phase material Vydac C4

Material for GHUR OF Vydac C4 (firm Vydac) consists of particles of silica gel, the surface of which are Sulkily chain. Department of the EPO protein contaminants based on differences in the strength of their hydrophobic interactions. Elution is carried out in a gradient of acetonitrile in dilute triperoxonane acid.

Preparative GHUR carried out using a column made of stainless steel (filled 2,8-3,2 l of silica gel Vydac C4). The eluate obtained with Hydroxyapatite Ultrogel chromatography, acidified by adding triperoxonane acid, and loaded into a column Packed with Vydac C4. For washing and elution using a gradient of acetonitrile in dilute triperoxonane acid. Fractions are collected and immediately neutralized phosphate buffer. Combine soderzhaschiesia faction, which meet the requirements of IPC (control of the production process).

d) Chromatography using DEAE-Sepharose

Material DEAE-Sepharose, Pharmacia) consists of diethylaminoethyl (DEAE) groups covalently associated with the surface of the granules separate. Binding of EPO with DEAE groups is mediated by ionic interactions. Acetonitrile and triperoxonane acid is passed through the column without delay. After these substances are washed, trace quantities of contaminants are removed by washing the column acetate buffer with low pH value. Then the column is washed with neutral phosphate buffer and EPO elute buffer with increased ionic strength.

The column is filled DEAE-Sepharose designed for chromatography with high flow rate. The column volume regulate in such a way as to ensure the loading of EPO is based 3-10 mg EPO/ml of gel. The column is washed with water and equilibrating buffer (phosphate sodium/potassium), Download the combined fractions obtained after elution using GHUR, and the column was washed with equilibrating buffer. After that, the column was washed with wash buffer (sodium acetate buffer), and then washed with equilibrating buffer. After that EPO elute from the column using an eluting buffer (sodium chloride, sodium phosphate/potassium) and collected in a single fraction with the under the main elution profile.

The eluate obtained from the column filled with DEAE-Sepharose, bring to a certain conductivity. The resulting substance for the preparation of a medicinal product is filtered under sterile conditions in vessels made of PTFE (Teflon®) and stored at -70°C.

Example 2: Pegylation of EPO using MPEG-QMS

EPO purified as described in example 1 method in serum-free medium (EPOsf)is homogeneous according to the evaluation using analytical methods and has a typical scheme isoforms, consisting of 8 isoforms. It has a specific biological activity 190000 IU/mg, as determined in the experience of using normocytic mice. Used PageLime reagent is a methoxy-PEG-QMS, i.e. the compound of formula II in which R denotes methyl; x is 3; and m denotes a number from 650 to 750 (from an average of 680, which corresponds to an average molecular weight of about 30 kDa).

The reaction paglierani

To 100 mg EPOsf (9,71 ml stock solution EPO concentration of 10.3 mg/ml, 548 mmol) is added 10 ml of 0.1 M potassium phosphate buffer, pH 7.5, containing 506 mg of methoxy-PEG-QMS with a molecular mass of 30 kDa (16.5 mmol) (obtained from the firm's Shearwater Polymers Inc., Huntsville, Alabama), and stirred for 2 h at room temperature (20-23°). The final protein concentration is 5 mg/ml and the ratio of protein: PEG-reagent composition is employed, 1:3. After 2 hours the reaction is stopped, bringing the pH to 4.5 with glacial acetic acid, and stored at -20°before cleaning.

Clean

1. A mixture of conjugates: Approximately 28 ml SP-SEPHAROSE FF (sulfopropyl cation-exchange resin) is loaded into a glass column type AMICON (2,2×7.5 cm) and the balance of 20 mm acetate buffer, pH 4.5, at a flow rate of 150 ml/hour, 6 ml reaction mixture containing 30 mg of protein was diluted 5 times equilibrating buffer and applied to the column. Needsomeone products are washed with buffer, and the adsorbed mixture of PEG-conjugate elute from the column using 0,M NaCl in equilibrating buffer. Still remaining in the column unmodified EPOsf elute 750 mm NaCl. Column re-balance the source buffer. Samples analyzed by SDS page-ordinator and determine the degree of their paglierani. It is established that the eluate obtained with 0,M NaCl, contains mono-and di - and trace amounts trippelironi species, while the eluate obtained through 750M NaCl, contains unmodified EPOsf. Di-PEG, Tonopah-EPOsf: Purified mixture of conjugates, elyuirovaniya from the column in the previous phase, dilute with buffer 4 times, re-make into a column and washed as described above. Di-PEG-EPOsf, Tonopah-EPOsf elute separately from the column with 0.1m NaCl and M NaCl, respectively. Hold e is a key using 750M NaCl, to be eluted entire remaining unmodified EPOsf.

Alternatively, the reaction mixture was diluted 5 times with acetate buffer and applied to a column Packed with SP-Sepharose (˜0.5 mg protein/ml gel). The column is washed and the adsorbed nanopeg-EPOsf, di-PEG-EPOsf and unmodified EPOsf elute according to the method described in the previous section.

Results

PEG-EPOsf synthesized by chemical conjugation with a linear PEG molecule with an average molecular mass of 30 kDa. PEG-EPOsf receive as a result of the reaction between the primary amine groups EPOsf and derived Succinimidyl ester butyric acid and PEG with a molecular mass of 30 kDa, leading to the formation of amide linkages.

The results are summarized in table 1. The purified mixture of conjugates contains mono - and di-PEG-EPOsf and does not contain unmodified EPOsf that is installed using an analysis using SDS page-ordinator. A mixture of conjugates is 23,4 mg or 78% of the original material. Separation using cation-exchange chromatography of mono - and di-PEG-EPOsf has allowed to establish that the ratio of mono - and di-PEG in the mixture of conjugates is about 1:1. After completion of the reaction the ratio of the individual components mono:di:unmodified EPOsf 40:38:20 (%). The total yield is practically quantitative.

Table 1

The generalization of the results by pegylation EPOsf
SampleProtein (mg)Output(%)
The reaction mixture30100
mono-to 12.040
di-11,438
unmodified6,020
a mixture of conjugates23,478

Example 3: Pegylation of EPO using MPEG-SEC

Different aliquot number EPOsf specified in example 2 is subjected to interaction with methoxy-PEG-SEC with a molecular mass of 30 kDa, Shearwater Polymers Inc., Huntsville, Alabama). The reaction is carried out at a ratio of protein: reagent 1:2 and apply the methods of purification described in example 2. Get mainly monopegylated types.

Example 4: Covalent binding tylnej groups with EPO

This example describes the results of determining the reaction conditions for covalent binding tylnej groups with EPO. To determine the conditions of different amounts of reagent containing blocked Tilney group, in this case, SATA or SATP (dissolved in DMSO to a concentration of 10 mg/ml), are added to a solution of EPO, in this case to 1 ml EPO with a concentration of 5 mg/ml in 10 mm is estate potassium, 50 mm NaCl, pH 7.3. The reaction mixture is stirred for about 30 min (25° (C) and the reaction stopped by adding 1M solution of lysine concentrations up to 1 mm. Excessive SATA and SATP is removed by dialysis in a counter 10 mm potassium phosphate, 50 mm NaCl and 2 mm add, pH of 6.2. The number of covalently linked with EPO tylnej groups after removal using hydroxylamine protective acetyl groups determined by photometric using dithiodipyridine according to the method described in Grasetti D.R. and Murray J.E. in: Appl. Biochem. Biotechnol. 119, p.41-49 (1967).

The table below presents data on the number tylnej groups, covalently linked to a molecule of EPO

The molar ratio of EPO: SATA or SATPThe number tylnej groups (in moles)/EPO (in moles)
EPO:SATA=1:31,5
EPO:SATA=1:52,4
EPO:SATA=1:63,2
EPO:SATP=1:31,3
EPO:SATP=1:42,5
EPO:SATP=1:63,7

Example 3: Modification of activated EPO with methoxy-PEG-maleimide

A) activation of the EPO:

100 mg of EPO obtained according to example 1 (190000 IU/mg, determined by analysis using normocytic mice), activate SATA (molar aspect] is the solution of EPO:SATA=1/5) according to the method described in example 2. The resulting EPO ("activated EPO"), bearing covalently linked blocked tirinya group, is separated from side products such as N-hydroxysuccinimide, or unreacted SATA by dialysis, according to the method described in example 1. Get solution containing 4.5 mg/ml of activated EPO in 10 mm potassium phosphate, 50 mm NaCl, 2 mm add, pH of 6.2.

B) Pegylation of activated EPO:

380 mg of methoxy-PEG-maleimide having the above ' preferred ' structure (MM 30000; firm Shearwater Polymers Inc., Hunstville, Alabama, USA), dissolved in the above solution containing 95 mg of activated EPO (4.5 mg/ml in 10 mm potassium phosphate, 50 mm NaCl, 2 mm add, pH 6,2), In the molar ratio of activated EPO and methoxy-PEG-maleimide in the solution is 1:4. Adding 1M aqueous solution of hydroxylamine to the above solution to achieve a concentration of 30 mm, a pH of 6.2, perform the unlocking covalently linked blocked tylnej groups of activated EPO. The formed activated EPO, in the reaction mixture in solution, is available tirinya (-SH) groups. Immediately after unlocking tylnej groups carry out the reaction mix between activated EPO, which now contains free tirinya (-SH) groups, and methoxy-P Is G-maleimido within 90 minutes (stirring, 25°). The reaction mix, stop adding to the reaction mixture to 0,2M aqueous solution of cysteine to a concentration of 2 mm. After 30 min the excess of free tylnej groups of activated EPO, which is not reacted with methoxy-PEG-maleimide block by adding 0.5m solution of N-methylmaleimide in DMSO to achieve a concentration of 5 mm. After 30 min the resulting reaction mixture already containing paglierani types of EPO, is subjected to dialysis in a counter 10 mm potassium phosphate, pH 7.5 over a period of time ≥15 p.m.

C) Cleaning paglierani species EPO

To highlight paglierani types of EPO from the reaction mixture is performed following the process of cleaning: 50 ml Q-Sepharose ff (high flow rate)-column balance 10 mm potassium phosphate, pH 7.5. Obtained in stage B), the reaction mixture was loaded into a column (flow rate: 3 volume of the column (s)/h). To separate unreacted methoxy-PEG-maleimide column was washed with 5 OK 10 mm potassium phosphate, pH 7.5. Paglierani types EPO separated by elution with increasing salt gradient in 5 OK buffer A (10 mm potassium phosphate, pH 7.5) and 5 OK buffer B (10 mm potassium phosphate, 500 mm NaCl, pH 7.5) at a flow rate of 3 OK/h Due to the presence of the gradient of NaCl at first suiryudan paglierani types of EPO (tri-, di - and monopegylated types EPO), and then naegeliana types of EPO. The fraction of luata, containing paglierani types of EPO (tri-, di - and monopegylated types EPO), are combined and filtered (filtration under sterile conditions using a filter with pore size 0.2 μm).

The content and purity of tri-, di - and monopegylated species EPO estimated at PAG-LTO-gels by staining Kumasi diamond blue (Laemmli, Nature, 227, 680-685 (1970)), and the protein concentration determined at 280 nm according to the law of beer-Lambert. The apparent molecular mass species EPO was determined using SDS page-ordinator electrophoresis, approximately 68 kDa (monopegylated types EPO), approximately 98 kDa (dipejirine types EPO) and approximately 128 kDa (three-Paglierani types EPO).

Additional separation of tri-, di - and monopegylated types of EPO can be done using chromatography, such as gel filtration (Superdex, 200 pg; firm Pharmacia).

Determination of biological activity in vivo of the eluate containing tri-, di - and monopegylated species, carried out according to the method described below.

Example 6: Activity in vivo paglinawan EPO was determined using analysis using normocytic mice

Biological analysis using normocytic mice known in this field (Pharm. Europa Spec. Issue Erythropoietin BRP Bio, 1997(2)), this method is described in the monograph on erythropoietin Ph. Eur. BRP. Samples diluted with BSA-STR. Normal ZV the global mice aged 7-15 weeks injected subcutaneously with 0.2 ml fractions EPO, containing naegeliana EPO or tri-, di - or monopegylated EPO obtained according to the method described in example 2 or 3. Within 6 days draw samples of blood by puncture of the tail vein and diluted so that 1 ml of solution dyed 0,15 µm acridine orange was present in 1 μl of blood. The application is 3-10 min. Number of reticulocytes determine microfluorometry using a flow cytometer, by analyzing the histogram of the red fluorescence. Reticulocyte count is given as absolute values (30000 analyzed blood cells). Presents data obtained in one day for groups, each consisting of 5 mice, and in mice take blood only once.

In another series of experiments, mice injected with a single dose remodelirovania EPO (25 ng EPO), a mixture of PEG(QMS)-EPO obtained in example 2 (10 ng conjugate), mono - and deparround EPO obtained in example 2 (10 ng conjugate), PEG(SEC)-EPO obtained in example 3 (10 ng conjugate) and buffer solution. The results are shown in table 2. The results show higher activity and prolonged time-life paglierani types of EPO, as evidenced by a significant increase in the number of reticulocytes and the shift of the maximum number of reticulocytes in the COI is whether the same dose/mouse (10 ng) compared with the dose of 25 ng unmodified EPO.

Table 2
EPO (unmodified)30 kDa SEC-PEGMono - 30 kDa QMSDi - 30 kDa QMSA mixture of conjugates of PEG-EPO QMSControl buffer
C1000139314119941328857
96 h500140615019261338697
120 h˜20011001182791944701
144 h˜0535607665660708

Example 7: a product, which consists mainly of Tonopah-EPO

The reaction paglierani

As the original product use 100 mg (5,48 of µmol) EPOsf in 100 mm potassium-phosphate buffer, pH 7.5, obtained according to example 1, to it add 329 mg (10,96 of µmol) reagent 30 kDa PEG-QMS, dissolved in 3 ml of 1 mm HCl. Add another 100 mm potassium phosphate buffer, pH 7.5, bringing the volume of the reaction mixture to 20 ml Final protein concentration is 5 mg/ml and the ratio of protein:PEG is 1:2. The reaction with the ect stirred for 2 h at ambient temperature (20-22° C). After 2 hours the reaction is stopped, bringing the pH to 4.5 with glacial acetic acid, and stored at -20°before cleaning.

Clean

Obtained in the previous phase of the reaction mixture is diluted in the ratio 1:5 10 mm sodium acetate, pH 4.5, and bring in 300 ml SP-Sepharose FF (sulfopropyl cation-exchange resin), loaded into the column size 4,2×19 cm Column pre-balance the same buffer. Coming out of the speakers products tested at a wavelength of 280 nm using a UV-monitor type Gilson and data is recorded by a recording device firms Kipp and Zonen. The column was washed with 300 ml or volume equal to the volume of 1 layer equilibrating buffer to remove excess reagents, by-products of the reaction and oligomeric PEG-EPO. Then rinse 100 mm NaCl, using a volume equal to the volume of 2 layers, in order to remove di-PEG EPO. Then elute nanopeg-EPO with 200 mm NaCl. During the process of elution of Tonopah-EPO first 50 ml of the peak protein is removed and Tonopah-EPO collected as fractions with a volume of 150 ml Unmodified EPOsf left on the column, elute 750 mm NaCl. All eluting buffers are prepared in equilibrating buffer. All erwerbende samples analyzed by SDS page-ordinator and using gel filtration high-resolution (SEC). Pool nanopeg-EPO obtained from fractions of 10 ml, which has no detectable quantities of unmodified EPOsf, then concentrate to obtain the concentration ˜4,5-7,5 mg/ml and subjected to filtration by means of dialysis (diafiltrate) in a counter buffer for storing, representing a 10 mm potassium phosphate, 100 mm NaCl, pH 7.5. Concentration/diafiltration carried out at ambient temperature, using a system of Millipore Labscale™ TFF System with membrane type Millipore Pellicon XL Biomax 50, which transmits products with a maximum molecular weight of 50 kDa. A concentrated nanopeg-EPO is sterilized by filtration and stored frozen at -20°C.

Pagelines approximately 75% EPOsf. After cleaning, the total output nanopeg-EPO, do not contain detectable quantities of unmodified EPOsf is ˜30%, the output dipag-EPO is approximately 25%. The rest of the protein is an oligomer and naegeliana EPOsf. Pool nanopeg-EPO obtained from the fraction by volume of 150 ml, contains about 90% of Tonopah-EPO and about 10% of depag-EPO.

Example 8: the Stability of EPO and paglinawan EPO in different compositions: analysis using DSC (differential scanning calorimetry)

It is widely known that the temperature of phase transition during heat denaturation was measured by the method of differential scanning calorimetry, is an important indicator is remotability proteins. Solutions erythropoietin or paglinawan erythropoietin concentration from 0.6 to 1.2 mg/ml in various buffers in the presence of stabilizers and without them analyze using calorimeter type Nano-DSC (firm Calorimetric Sciences Corporation, Utah, USA) at a heating rate of 2 K/min to Increase the phase transition temperature indicates an increase in thermostability of the protein. The measured temperature values should not be understood as absolute values, they are likely to characterize the differences between stabilnosti separate compositions.

To determine the optimum pH of the composition to study the dependence of thermal denaturation paglinawan erythropoietin on pH in the range from 4 to 9. The protein samples analyzed in 30 mm Na2HPO4, 30 mm sodium citrate, 30 mm borate. Figure 3 shows that the maximum temperature of the phase transition goes on a plateau in the pH range from about 6 to about 9 and sharply decreases at pH values less than 5.5. This suggests that the optimal pH value for maximum thermal stability in excess of 5.5 (figure 3).

To study the influence of ionic strength determine the dependence of thermal denaturation on the concentration of phosphate. The data shown in figure 4, indicate that thermal stability increases with increasing ionic siliconosis.

The influence of the buffer substance is also examined using DSC. The data shown in figure 5, indicate that the most appropriate buffers or additives to provide high thermal stability are sulfate, citrate or phosphate. Glycine, which is used as the buffer used in the present compositions (see above), is not optimal for this purpose.

The data shown in Fig.6 indicate that sulfate is acceptable buffer/additive at low pH values (for example, when the pH of 6.2), while phosphate at pH 6,2 less effective than at pH 7.5. This suggests that sulfate helps to maintain thermal stability at a high level even at low pH values. This discovery allows you to create a composition having a pH value of from 6.0 to 6.5, which does not result in a significant loss of thermal stability of erythropoietin.

Example 9: Aggregation of EPO and PEG-EPO in terms of thermal stress: analysis by SDS page-ordinator (gel-electrophoresis in polyacrylamide gel with sodium dodecyl sulfate)

To explore the impact of heat stress on protein erythropoietin samples of different compositions are subjected to thermal stress (20 min at 80° (C) and analyzed by gel-electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS page-ordinator) vosstanavlivajusa (in the sample buffer is present DTT) and non (in sample buffer lacking DTT) conditions. This method allows to detect the formation of covalently linked aggregates. As mentioned above, the formation of aggregates is one of the main reasons for the degradation of proteins, and therefore it is necessary that it has not taken place in the pharmaceutical compositions of proteins. With high probability we can assume that the aggregates that can be detected in the absence of reducing agent (such as DTT), and therefore cannot be detected in the presence of a reducing agent, there are under stress due to the formation of incorrect disulfide bonds in the oxidation reaction. Figure 5 presents the dependence of the degree of aggregation from pH under conditions of heat stress. The results of this experiment clearly indicate that the formation of aggregates decreases at pH values below 6.5. The higher the pH value, the greater the number of units. Most of the resulting aggregates can be recovered by processing samples of the reducing agent in the implementation of the PAG-ordinator, suggesting that a large part of the aggregates formed under heat stress, is linked by a disulfide bridges dimers, oligomers and aggregates of higher order. Taken together, these data indicate that formation of aggregates largely preventable, is if the pH value of the composition be maintained at 6.5 or lower.

7: dependence of aggregation of PEG-EPO from pH. Presents the results of the analysis using SDS page-ordinator samples of PEG-EPO after heat stress (described above). Proteins stained with silver. Lane 1: standard molecular weight. Lane 2: pH 5. Band 3: pH 5, after recovery. Band 4: pH 6. Band 5: pH 6, after recovery. Band 6: pH 6.5. Band 7: pH 6.5, after recovery. Band 8: pH 7. Band 9: pH 7, after recovery. Band 10: PEG-EPO not subjected to stress.

The formation of aggregates can be prevented by using antioxidants. On Fig demonstrated that the application of 1 mg/ml acetylcysteine as an antioxidant prevents the formation of aggregates under conditions of heat stress. Therefore, to prevent the formation of aggregates in terms of thermal stress can be applied antioxidants, such as NAC, at low pH values, for example, at pH of 6.2.

6: Formation of aggregates PEG-EPO can be prevented by maintaining the pH value at the level of 6.2 and/or applying acetylcysteine. Presents the results of the analysis using SDS page-ordinator samples of PEG-EPO after heat stress (described above). Proteins stained with silver. Lane 1: PEG-EPO not subjected to stress. Lane 2: pH 7.5, after stress. Band 3: pH of 6.2, after stress. Band 4: pH of 6.2,after stress, after recovery. Band 5: pH 7.5, 1 mg/ml acetylcysteine, after stress. Band 6: pH 7.5, 1 mg/ml acetylcysteine, after stress and after recovery.

Example 10 Stability of PEG-EPO in various compositions at 4. 25, 30 and 40°

Targeted EPO in various compositions incubated at several temperatures. At certain points in time selected samples and evaluate stability using liquid chromatography high-resolution reversed-phase (RP-GHUR), gel filtration, high-resolution (SEC) and electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS page-ordinator). Table 3 presents a comparison of the stability of PEG-EPO in various compositions after storage at specified temperatures. These data clearly indicate the superior properties presented in this description of the songs from the viewpoint of the recovery of the protein and formation of aggregates.

Table 3

The stability of PEG-EPO in various compositions at several specified temperature
% recovery after 6 months of storage atDiscovered aggregation at 30° (+/- )
Composition*PEG-EPO (µg/ml)4#x000B0; 25°30°40°
And109291the concentration is39the concentration is
B5097989778-
In5095797952+
D5010310210087-
And1009697the concentration is50the concentration is
B40010110110177-
In400100949056+
G40098969373-
D400999810066-
* in the compositions include:

composition: 10 mm sodium phosphate, 100 mm sodium chloride, pH 7.5;

composition B: 10 mm sodium phosphate, 40 mm sodium sulfate, 3% wt./about. mannitol, PH 6,2;

the song In: 10 mm sodium phosphate, 100 mm NaCl, pH 7.0;

composition: 10 mm sodium phosphate, 120 mm sodium sulfate, pH 6,2;

composition D: 40 mm arginine, 30 mm sodium sulfate, 3% mannitol, 1 mm CaCl2pH of 6.2.

Example 11: the Optimal composition used to prevent the oxidation of the methionine at position 54 (methionin) protein EPO when using methionine as an antioxidant

Various compositions, which include targeted EPO incubated at several temperatures. After 6 months take samples and determine the degree of oxidation methionine according to the method described below. In General, the method consists in the following. Samples EPO treated with endoproteinase LysC. The resulting peptides separated using chromatography with reversed phase. Calculate the ratio of the content of oxidized peptide T8 (bearing oxidized methionin) and content of the peptide T8 (carrying oxygenated methionin). The data are summarized in table 4.

Table 4

Oxidation methionine protein EPO in various compositions after 6 months storage at specified temperatures
% oxidized methionine
Composition*PEG-EPO (µg/ml)25&#HWS 30°40°
And4002,0015,8924,8937,89
B4002,336,5512,8830,24
In400of 2.512,955,9914.4V
And50lower than the 5.3721,3630,6248,05
B503,4413,3816,5930,83
In50to 4.415,5210,0115.62 wide
* in the compositions include the following compounds

composition: 10 mm sodium phosphate, 100 mm sodium chloride, pH 7.0;

composition B: 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2.

composition: 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, 1 mm methionine, pH of 6.2.

The data presented clearly indicate that site Occitania composition of the present invention (10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH 6,2) is superior in the degree of oxidation methionine other songs, such as 10 mm sodium phosphate, 100 mm NaCl, pH 7.0. Added the e to the composition of 1 or 10 mm methionine expression reduces oxidation methionine. Therefore, methionine acts as an antioxidant and stabilizes the EPO.

Example 12: the Content of sialic acid in samples of PEG-EPO for different compositions

To assess the safety of carbohydrate structures of glycoproteins PEG-EPO carried out using standard methods of analysis of the content of sialic acid in samples of PEG-EPO after storage for 6 months at different temperatures. The data obtained indicate that the storage of the above-described new compositions containing the protein has no adverse effect on the integrity of the carbohydrate structure (Fig.9).

Example 13: the Biological activity paglinawan EPO after storage for extended periods of time at elevated temperatures

To ensure that the storage of PEG-EPO in the composition containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH 6,2 not have a negative impact on biological activity in vivo, conduct standard research activity of EPO in mice (see example 6). For samples of PEG-EPO samples, which were stored at the indicated temperatures on the graph for 6 months in the composition containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH 6,2, during storage at 4, 25 and 30°With no loss of activity in vivo no comparison what about the fresh sample, used as a standard (figure 10).

Example 14: the content of the units of PEG-EPO after storage at elevated temperatures for 6 months

For the content analysis of aggregates in the samples of PEG-EPO after storage for 6 months at an elevated temperature in the composition containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, pH of 6.2, taking samples and perform the analysis using gel filtration.

At 4, 25 and 30°not detected the presence of aggregates, which indicates the stability paglinawan EPO, part of the above-described compositions. Figure 11 shows a comparison of chromatograms obtained by gel filtration.

1. Liquid pharmaceutical composition comprising a protein targeted erythropoietin, which represents a conjugate that includes the glycoprotein erythropoietin having at least one free amino group and having the biological activity in vivo, leading to increase production of reticulocytes and red blood cells by the bone marrow cells, and selected from the group comprising human erythropoietin and its analogues, which have the sequence of human erythropoietin modified d is the deliverance from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; this glycoprotein covalently linked to "n" poly(etilenglikolevye) groups of the formula-CO-(CH2)x-(Och2CH2)m-OR, where the radical WITH each poly(etilenglikole) group forms an amide bond with one of these amino groups; R represents (ness.)alkyl; x is 2 or 3; m is a number from about 450 to about 900; n is 1-3; and n and m are chosen so that the molecular weight of the conjugate minus the molecular weight of the protein erythropoietin ranged from 20 to 100 kDa, multiply charged inorganic anion in a pharmaceutically acceptable buffer suitable for maintaining the pH in the solution in the range from approximately 5.5 to approximately 7,0, and optionally one or more pharmaceutically acceptable excipients, and the specified liquid composition is stable at room temperature.

2. The composition according to claim 1, in which the protein erythropoietin represented by the formula:

where m, n, x and R have the above meanings and R represents the residue of a glycoprotein without n aminogroup(p), which(s) form the amide(s) relationship(s) with poly(etilenglikole(s)) group(s).

3. The composition according to claim 1, which is an aqueous solution.

4. The composition according to claims 1 to 3, which represents an isotonic solution.

5. The composition according to claims 1 to 4, where Ani is n represents a multiply charged anion of a strong inorganic acid.

6. Composition according to claims 1-5, where the anion is chosen from the group comprising sulfate, citrate or phosphate.

7. Composition according to claims 1-6, where the anion is a sulfate.

8. The composition according to claim 7, containing from 10 to 200 mmol/l of sulfate.

9. Composition according to claims 1 to 8, wherein the pH value is from about 5.8 to 6.7.

10. The composition according to claim 9, where the pH is from 6.0 to 6.5.

11. The composition of claim 10, where the pH is about 6.2.

12. The composition according to claims 1 to 11, where the buffer is chosen from the group comprising phosphate buffer or arginine N2SO4/Na2SO4buffer.

13. The composition according to item 12, where the buffer is 10 to 50 millimolar phosphate buffer.

14. The composition according to claims 1 to 13, where the composition comprises one or more pharmaceutically acceptable excipients.

15. The composition according to 14, where one or more of pharmaceutically acceptable excipients are selected from the group comprising pharmaceutically acceptable salts, diluents, solvents and preservatives.

16. The composition according to PP and 15, where the pharmaceutically acceptable excipients are selected from the group comprising agents to maintain toychest, polyols, antioxidants and nonionic surfactants.

17. The composition according to p-16, where the pharmaceutically acceptable excipient is a polyol.

18. The composition according to 17, where the polyol is chosen from g is uppy, including mannitol, sorbitol, glycerol, trehalose and sucrose.

19. The composition according to p, where the polyol is a mannitol.

20. The composition according to PP-19, containing the antioxidant.

21. The composition according to claim 20, where the antioxidant is a methionine.

22. The composition according to p-21, containing up to 1 mmole/l CaCl2.

23. The composition according to PP-22, where the nonionic surfactant is a Polysorbate 80, Polysorbate 20 or pluronic F68.

24. The composition according to item 23, where the nonionic surfactant is pluronic F68.

25. The composition according to item 23 or 24, where the composition contains up to 1% (wt./about.) nonionic surfactants.

26. Composition according to any one of p-25, where the composition contains up to 0.1% (wt./about.) nonionic surfactants.

27. The composition according to claims 1 to 26, where the protein erythropoietin is human erythropoietin.

28. The composition according to item 27, where the protein erythropoietin Express through the endogenous gene activation.

29. The composition according to PP-28, where the protein erythropoietin has the amino acid sequence represented in SEQ ID NO:1 or SEQ ID NO:2.

30. The composition according to p-29, where the protein has the sequence of human erythropoietin modified by adding from 1 to 6 glycosylation sites.

31. Composition p is p-29, where the protein erythropoietin has the sequence of human erythropoietin modified by a modification selected from the group including:

Asn30Thr32,

Asn51Thr53,

Asn57Thr59,

Asn69,

Asn69Thr71,

Ser68Asn69Thr71,

Val87Asn88Thr90,

Ser87Asn88Thr90,

Ser87Asn88Gly89Thr90,

Ser87Asn88Thr90Thr92,

Ser87Asn88Thr90Ala162,

Asn69Thr71Ser87Asn88Thr90,

Asn30Thr32Val87Asn88Thr90,

Asn89lle90Thr91,

Ser87Asn89lle90Thr91,

Asn136Thr138,

Asn138Thr140,

Thr125and

Pro124Thr125.

32. The composition according to p-31, where the sequence of human erythropoietin modified by the rearrangement of at least one glycosylation site.

33. The composition according to p, where the rearrangement involves the deletion of any of the N-linked glycosylation sites in human erythropoietin and the addition of N-linked glycosylation site at position 88 of the sequence of human erythropoietin

34. The composition according to p, where the glycoprotein has the sequence of human erythropoietin modified by a modification selected from the group including:

Gln24Ser87Asn88Thr90,

Gln38Ser87Asn88Thr90and

Gln83Ser87Asn88Thr90.

35. The composition according to p-34, where specified in any of paragraphs protein erythropoietin is paglierani.

36. The composition according to claim 2, where in the formula (I) x is 2, m denotes a number from 650 to 750, n is 1 and R is methyl.

37. Liquid pharmaceutical composition comprising a protein, where the protein erythropoietin is a conjugate, the conjugate contains glycoprotein erythropoietin having at least one free amino group and having the biological activity in vivo, leading to increased production of reticulocytes and red blood cells by the bone marrow cells, and which are selected from the group comprising human erythropoietin and its analogues having the primary structure of human erythropoietin modified by the addition of 1 to 6 glycosylation sites; where the glycoprotein covalently associated with one-three (ness.)alkoxyl(etilenglikolevye) groups, each poly(etilenglikolya) group covalently linked to the glycoprotein using the linker of the formula-C(O)-X-S--with fragment C(O) linker, resulting in an amide bond with one of the amine groups. X represents -(CH2)k- or-CH2(O-CH2-CH2)k-, k is 1 to 10, Y represents

or

the average molecular weight of each poly(etilenglikole) fragment is from about 20 to about 40 kDa, and the molecular mass of the conjugate is from about 51 to about 175 kDa.

38. The composition according to clause 37, where the protein erythropoietin is a conjugate of the formula:

where n denotes an integer from 1 to 3; m represents an integer from 450 to 900; R is (ness.)alkyl; X represents -(CH2)k- or-CH2(O-CH2-CH2)kand R denotes the residue of the glycoprotein erythropoietin without the amino group or groups, forming an amide bond with X.

39. The composition according to clause 37, where the protein erythropoietin is a conjugate of the formula:

where n denotes an integer from 1 to 3; m represents an integer from 450 to 900; R is (ness.)alkyl; X represents -(CH2)k- or-CH2(O-CH2-CH2)kand R denotes the residue of the glycoprotein of eritro Atina without the amino group or groups, forming an amide bond with X.

40. The composition according to claims 1 to 39, containing from 10 to 10,000 μg protein erythropoietin in ml.

41. The composition according to claims 1 to 40, containing from 10 to 10,000 μg protein erythropoietin per ml, 10-200 mmol/l of sulfate and 10-50 mmol/l phosphate, pH 6,0-6,5.

42. The composition according to claims 1 to 41, containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol and 1 mm methionine, pH of 6.2.

43. The composition according to PP and 42 containing up to 20 mm methionine, 1-5% (wt./about.) polyol, up to 0.1% (wt./about.) pluronic F68 and optional up to 1 mm CaCl2.

44. The composition according to p-43 containing 10-10000 mg of protein erythropoietin per ml, 40 mmol/l of sulfate, 10 mmol/l phosphate, 3% (wt./about.) mannitol, 10 mm methionine, 0.01 percent (wt./about.) pluronic F68, pH of 6.2.

45. The composition according to claims 1 to 40, containing 10-10000 mg of protein erythropoietin per ml, 10-100 mmol/l NaCl, 10 to 50 mmol/l phosphate, pH 6,0-7,0, optional 1-5% polyol.

46. The composition according to item 45, containing up to 20 mm methionine, 0.1% pluronic F68 and optional 7.5 mmol/l CaCl2.

47. The composition according to item 45 or 46, containing 10-10000 mg of protein erythropoietin per ml, 100 mmol/l NaCl, 10 mm methionine, 0.01% of pluronic F68 and 10 mmol/l phosphate, pH 7.0.

48. The composition according to claims 1 to 40, containing 10-10000 mg of protein erythropoietin per ml, 10-50 mmol/l arginine, pH 6,0-6,5, 10-100 mmol/l of sodium sulfate.

49. The composition according to p containing up to 20 mm methionine, 0.1% pluronic F68, neoba is consequently up to 1 mmol/l CaCl 2and optional 1-5% polyol.

50. The composition according to p or 49 containing 10-10000 mg of protein erythropoietin per ml, 40 mmol/l arginine, pH 6,2, 30 mmol/l of sodium sulfate, 3% mannitol, 10 mm methionine, 0.01% of pluronic F68, optional 1 mmol/l CaCl2.

51. The composition according to claims 1 to 40, containing 25-2500 µg/ml erythropoietin, and

a) 10 mm phosphate sodium/potassium, 100 mm NaCl, pH 7.0 or

b) 10 mm sodium phosphate, 120 mm sodium sulfate, pH of 6.2 or

in) 10 mm sodium phosphate, 40 mm sodium sulfate, 3% mannitol, pH of 6.2 or

g) 10 mm sodium phosphate, 40 mm sodium sulfate, 3% mannitol, 10 mm methionine, 0.01% of pluronic F68, pH of 6.2 or

d) 40 mm arginine, 30 mm sodium sulfate, 3% mannitol, pH of 6.2 or

e) 40 mm arginine, 30 mm sodium sulfate, 3% mannitol, pH of 6.2, 0,01% pluronic F68, pH of 6.2.

52. The composition according to claims 1 to 51, where the amount of erythropoietin is 50, 100, 400, 800 or 2500 µg/ml.

53. The composition according to § 51, containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% mannitol, 10 mm methionine, 0.01% of pluronic F68, pH of 6.2.

54. The composition according to item 43, containing 10 mm sodium phosphate, 40 mm sodium sulfate, 3% (wt./about.) mannitol, 1 mm methionine, pH of 6.2.

55. The composition according to paragraph 52, containing 40 mm arginine, 30 mm sodium sulfate, 3% mannitol, 10 mm methionine, 0.01% of pluronic F68, pH of 6.2.

56. Composition according to any one of claims 1 to 55, designed to produce medicines cured for who I am and disease prevention, associated with anemia in patients with chronic renal failure (CRF)patients with AIDS and/or cancer patients undergoing chemotherapy.

57. A method of obtaining a composition according to any one of claims 1 to 55, providing a mixture of protein erythropoietin with a solution containing carries many negative charges on the anion, and optionally one or more pharmaceutically acceptable excipients.

58. Method for the treatment and prevention of diseases involving anemia in patients with chronic renal failure (CRF), AIDS patients and cancer patients undergoing chemotherapy to which the stages of introduction to the patient a composition according to any one of claims 1 to 55.

59. Device for local and systemic continuous injection of a composition according to any one of claims 1 to 55, selected from the group consisting of an implant, injection, and inhalation devices.



 

Same patents:

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a medicinal agent used in prophylaxis and treatment of asiderotic anemia. The preparation comprises components in their following ratio: ferbitol, 80-99%, and gelatin, 1-20%. Invention provides reducing toxicity of the prepared preparation, improvement of its biological activity that results to enhancing therapeutic effectiveness.

EFFECT: enhanced and valuable medicinal properties of agent.

6 ex

FIELD: medicine, pharmacology.

SUBSTANCE: agent for stimulation of erythropoiesis and elimination of iron deficiency comprises hemoglobin obtained from animal blood containing methemoglobin additionally. The ratio of hemoglobin and methemoglobin in the agent = (25-75)-(75-25) wt.-%. The agent shows the expressed heme-stimulating effect. The agent can be used in producing medicinal agents and biologically active supplements.

EFFECT: valuable medicinal properties of agent.

4 dwg

FIELD: medicine, veterinary science, pharmacy.

SUBSTANCE: invention relates to the iron-containing preparation "Ferral". Iron-containing preparation "Ferral" comprises iron in bivalent form - hydroxy-ferrous oxide in the concentration 2-9%. Invention provides administration of the preparation in amount practically by 10 times less as compared with administration of the known preparations that shows the sufficient effect and doesn't cause over dosing.

EFFECT: improved and valuable properties of preparation.

3 tbl, 4 ex

FIELD: medicine, immunology.

SUBSTANCE: invention relates to anemia treatment in infants with immunodeficiency condition. Claimed method includes alternation of tonsilgon H administration in dose of 25 drops 3 times per day for 2 weeks and IRS-19 in one dose in each nasal passage 2 times per day for 2 weeks during 3 months. Course is carried out 2 times per year in spring and autumn for two years on end. In addition to abovementioned preparations 1 ml of tymogen is administered intramuscularly 2 times per week, 2 ml of 12.5 % cycloferon is administered intramuscularly 1 time per week, 2 ml of erytrostim is administered percutaneously 1 time per week during 3 months 2 times per year in spring and autumn for two years on end.

EFFECT: method for effective recovery of red blood shoot and immunodeficiency immunopoesis.

1 tbl, 1 ex

FIELD: medicine, pediatrics, hematology.

SUBSTANCE: the present innovation could be applied in treating surgical and oncological diseases in children during treating and preventing anemia of different etiology. One should introduce ceruloplasmin at daily single dosage ranged 50-200 mg depending upon child's age against 100-200 ml 0.9%-sodiumchloride solution intravenously by drops at the rate of 40-50 drops/min. Moreover, for preventing and/or treating posthemorrhagic anemia in case of surgical operations one should introduce ceruloplasmin during 2 d before surgical operation, intraoperationally, and for 2-10 d after operation. For preventing and/or treating anemia in case of purulent-septic diseases ceruloplasmin should be introduced during chemotherapeutic days during the whole period of the course conducted, for preventing and treating radiation anemia at the background of radiation therapy ceruloplasmin should be injected once weekly during the whole period of radiotherapy course, for preventing and treating toxic and radiation anemia at the background of chemoradiation therapy ceruloplasmin should be introduced once weekly on the day of chemotherapy during the whole course of chemoradiation treatment. The innovation enables to avoid inflows of erythrocytic mass and donor's blood along with shortened number of procedures on introduction of ceruloplasmin and 4-times decrease in the risk for the development of severe anemia in children due to matching peculiar mode for ceruloplasmin introduction.

EFFECT: higher efficiency of therapy and prophylaxis.

4 cl, 5 ex

FIELD: agriculture, animal husbandry, fur farming.

SUBSTANCE: invention relates to preparations used in prophylaxis and treatment of domestic and agricultural animals and for maintaining trace elements composition of fodders. The preparation allows balancing the nutrition diet for animals with the optimal ratio of trace elements providing prophylaxis of many diseases. Proposed preparation comprising complex of iron, manganese, copper, cobalt, selenium and zinc with ethylenediamine-N,N1-disuccinic acid disodium or dipotassium salt and water comprises additionally iodine in the following ratio of components: ethylenediamine-N,N1-disuccinic acid disodium or dipotassium salt, 15-25; iron (III), 1.5-5.5; manganese (II), 0.25-3.0; copper (II), 0.12-0.55; cobalt (II), from above 0.05 to 0.3; zinc (II), 0.05-1.5; selenium (IV), from above 0.03 to 0.06; iodine (I), 0.01-0.08; water, the balance.

EFFECT: improved and valuable properties of preparation.

1 cl, 4 tbl

FIELD: medicine.

SUBSTANCE: invention proposes an agent for correction of disturbances in production of cytokines and markers of activation of immune system cells in chronic iron-deficient anemia. Agent represents Sorbifer Durules known early as preparation used in treatment of iron-deficient anemia. Invention provides recovering the level of anti-inflammatory cytokines (IL-1, IL-6, IL-8, FNO-α, INF-α and INF-γ) and normalization of cellular immunity by indices of markers CD-25, CD-71 and CD-34.

EFFECT: valuable medicinal properties of agent.

2 tbl, 1 ex

FIELD: veterinary.

SUBSTANCE: agent is based on complex compound of colloidal iron liganded by polyoxymethylene-lauric alcohol and contains following components, mg: colloidal iron 50.0-60.0, lauric alcohol 0.1-0.3, copper 0.1-0.3, zinc 0.1-0.3, cobalt 0.1-0.3, selenium 0.001-0.005, and water to 1000. Agent can further contain 30.0-50.0 mg glucose and 0.1-0.6 mg polyvinyl alcohol.

EFFECT: enabled therapeutical and preventive efficiency at low toxicity, the same dispersity, and high stability upon storage.

3 tbl, 7 ex

FIELD: pharmaceutics, veterinary science.

SUBSTANCE: the present innovation deals with preventing and treating hypomicroelementosis in different farm and domestic animals, furred animals, and, also, for enhancing the growth in animals, and treating a number of specific diseases and, also, for maintaining microelemental composition of feedstuffs. The suggested preparation includes chelated complex of iron, manganese, zinc, copper, cobalt, selenium and iodine with organic ligand of complexone type and water. According to the innovation as a chelation ligand it contains trisodium salt of methionine succinic (α-amino-γ-methylthiobutyric-N-succinic) acid at a certain ratio of components. The innovation provides to obtain preparation in soluble form being capable to be well digested by the animal.

EFFECT: higher efficiency.

FIELD: medicine, pharmacology.

SUBSTANCE: invention relates to method for assay of optimized regimens in dosing erythropoietin (EOR). Invention proposes a system for election of one or some regimens in dosing by using pharmacokinetic/pharmacodynamic (PK/PD) as a model for assay of PK/PD pattern regimens. Then method involves selection of such regimen that provides the serum concentration of EOR exceeding its before administration for 5-30 days between administrations of EOR. Invention provides optimal applying EOR for correction of blood indices changes and treatment of diseases associated with them.

EFFECT: improved and valuable modeling method.

50 cl, 81 dwg, 2 tbl, 8 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a cycloferon-containing medicinal agent used in prophylaxis and treatment of influenza and acute respiratory diseases, herpetic infection, chronic viral hepatitis B and C and prophylaxis of oncological diseases. Agent comprises a physiological solution in the following ratio of components: 10-29 - 10-2 mg of cycloferon in 1 ml of physiological solution. Also, invention relates to a method for preparing this medicinal agent. Proposed medicinal agent enhances antiviral and antitumor activity of natural killers by 1.8-fold, not less.

EFFECT: improved preparing and using method, valuable medicinal properties of agent.

7 cl, 1 dwg, 19 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to new medicinal agents for parenteral using based on an antidepressant agent pirlindole salts. Agent comprises 2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino[3,2,1-j,k]carbazole (pirlindole) sulfonate salts as an active substance, such as methane sulfonate or benzene sulfonate. The composition for intravenous or intramuscular injection comprises additionally citric acid and water taken in the definite ratios. Invention provides preparing an agent possessing good tolerance, low toxicity and resistance in storage.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agent.

2 cl, 1 tbl, 4 ex

FIELD: pharmacy.

SUBSTANCE: invention proposes a liquid pharmaceutical composition containing nicotine in any form for administration into the mouth cavity and alkalinized with a buffer and/or by regulation of pH value. Administration is carried out preferably by spraying and the most preferably by sublingual spraying. Also, invention relates to a method for preparing the indicated composition. Use of indicated composition in therapy, such as therapy for treatment of addiction to tobacco.

EFFECT: valuable pharmaceutical properties of composition.

51 cl, 11 ex

FIELD: medicine.

SUBSTANCE: invention relates to microemulsion preconcentrate comprising active ingredient, oil, surfactant and hydrophilic solvent selected from group containing propylene glycol diacetate, propylene glycol acetate and salts thereof. Microemulsion preconcentrate optionally contains as active ingredient not only hydrophobic compounds but also hydrophilic compounds and proteins.

EFFECT: microemulsion preconcentrate of improved quality.

6 cl, 6 tbl, 3 dwg, 14 ex

FIELD: medicine.

SUBSTANCE: claimed viscoelastic based on 1.5-2.5 % methylcellulose contains additionally 0.0125-0.025 % dipivephrin hydrochloride solution and 0.0005-0.01 % of benzalconium chloride as stabilizer.

EFFECT: viscoelastic for prevention of eye tissues during intraocular interferences.

2 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention deals with creating pharmaceutical compositions containing bile acids and methods of treatment using these compositions. For this purpose, invention provides transparent aqueous solution containing bile acid or its compounds, polysaccharide, and water. Amounts of the two formers are selected so that they remain in solution at all values within selected pH range. Administration of such a composition increases level of enterohepatic bile acid in body, including blood, and also increases absorption of bile acid. Addition of various drug to composition allows effective treatment of corresponding diseases.

EFFECT: improved transportation to body tissues of bile acids both as individual therapeutic agents and as drug forms.

47 cl, 11 dwg, 16 tbl, 18 ex

FIELD: medicine, ophthalmology, chemico-pharmaceutical industry.

SUBSTANCE: the suggested pharmaceutical composition is indicated for local application and contains an inhibitor angiotensin converting enzyme as an active substance and target additions, moreover, the content of active substance corresponds to about 1-20 mg/ml. The composition suggested could be designed as eye drops, spay, gels, solution for local injections. As target additions one should apply water that contains a buffer agent, an isotonic mixtures, a conservant and a prolongator. Additionally, this composition contains preparations chosen out of the following groups: antibiotics, macro- and microelements, vitamins, adrenoblocking agents. The innovation provides anti-ischemic action, improves reparative processes and accelerates the processes of healing.

EFFECT: higher efficiency.

3 cl, 7 ex

FIELD: veterinary medicine.

SUBSTANCE: composition comprises solution for per os introducible to animals. The solution contains Phenasal and additives. Phenasal is applied as solution of its salt in aprotonic bipolar hydroxyl-containing solvents and bases.

EFFECT: high therapeutic activity; reduced Phenasal consumption; enhanced effectiveness of treatment.

4 cl, 2 tbl

Haemostatic agent // 2275201

FIELD: pharmaceutical industry, in particular haemostatic agent.

SUBSTANCE: claimed agent represents aqueous solution, containing 2-150 g of zinc polyacrylate, 10-100 g sodium alginate and 1-30 of acetic acid in 1 l of distilled water. Agent of present invention being in contact with blood and tissues provides film formation 30 s.

EFFECT: haemostatic agent of improves quality.

FIELD: pharmaceutical industry, in particular agent for treatment of joint diseases in form of injections.

SUBSTANCE: claimed agent contains glucoseamine salt, preservative, non-ionic surfactant, in particular oleic acid and polyoxyethylated sorbitan monoether, and water.

EFFECT: agent for treatment of joint diseases of improved effectiveness combining chondroprotevtive action with high pharmacokinetic properties and biological availability.

3 cl, 3 tbl, 4 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: the suggested ophthalmic composition contains ketothiphen salt, nonionic agent to regulate tonicity in the quantity that total tonicity of composition should correspond to osmolarity of 210-290 mOsm, a conserving agent, not obligatory, either acid or foundation to achieve pH value being slightly acid and water, moreover, if the mentioned conserving agent is not available, the concentration of ketothiphen salt should correspond to 0.01-0.04 weight%, or if the mentioned conserving agent is available, the concentration of ketothiphen salt should correspond to 0.01-0.03 weight%. As ketothiphen salt the suggested composition contains ketothiphen fumarate; tonicity should be controlled with nonionic agent - glycerol.

EFFECT: more prolonged action of the composition suggested.

15 cl, 2 ex

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