Liquid composition of factor vii polypeptides

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly liquid composition of factor VII polypeptides. Invention claims liquid aqueous composition including (i) factor VII polypeptide, (ii) substance suitable for pH maintenance approximately within 4.0 to 8.0, (iii) substance selected out of list including calcium salt, magnesium salt or their mix, where (iii) concentration comprises at least 15 mM, and antioxidation agent.

EFFECT: enhanced stability.

33 cl, 7 ex, 1 dwg

 

The present invention relates to liquid compositions containing the factor VII polypeptides, and to methods of producing such compositions. More specifically, this invention relates to liquid compositions stabilized against chemical and/or physical destruction.

Identified a number of factors involved in the blood clotting process, including factor VII (FVII) is a glycoprotein plasma. Hemostasis is initiated by formation of a complex between tissue factor (TF)that appear in the blood after injury of the vessel wall, and FVIIa, which is present in the bloodstream in an amount corresponding to about 1% by weight of the total protein FVII. FVII exists in the plasma, mainly in the form of single-stranded zymogen, which is cleaved by FXa to activated double-stranded form FVIIa. As prognosticheskogo agent developed recombinant activated factor VIIa (rFVIIa). The introduction of rFVIIa causes a rapid and highly efficient prohemostatic reaction in subjects with hemophilia bleeding, which cannot be treated by other products of coagulation factors due to the formation of antibodies. Also FVIIa can successfully treat bleeding in subjects with deficiency of factor VII or subjects with normal clotting system, but experiencing excessive bleeding.

It is desirable to have the forms for the administration of factor VIIa, suita is included for storage and for delivery. Ideally, a drug store and injected in liquid form. On the other hand, drug lyophilized, i.e. dried by freezing, and then restored by adding a suitable diluent immediately before use to the patient. Ideally, the drug has sufficient stability to be maintained during long-term storage, i.e. more than six months.

The decision to store whether the finished medicinal product in the form of liquid or dry it by freezing, as a rule, is based on the stability of protein drugs in such forms. On the stability of the protein, among others, can be influenced by factors such as ionic strength, pH, temperature, repeated cycles of freezing/thawing and effect of effort shift. The active protein can be lost due to physical types of instability, including denaturation and aggregation (formation of both soluble and insoluble aggregates), as well as the chemical forms of instability, including, for example, hydrolysis, deliciouse and oxidation, as several factors. For an overall view of stability of pharmaceuticals, see, for example, Manning et al., Pharmaceutical Research, 6:903-918 (1989).

Although there are instances of instability of proteins is well known, it is impossible to predict h the local problems of instability of a specific protein. Any type of instability can lead to the formation side of the protein product of or derived from a reduced activity, increased toxicity and/or increased immunogenicity. Indeed, the deposition of protein can lead to thrombosis, the heterogeneity of the dosage form and quantity, as well as to clogging of the syringe. In addition, post-translational modifications such as, for example, gamma-carboxylation of certain glutamic acid residues in the N-end and the addition of carbohydrate side chains, creating potential sites that may be susceptible to modification after storage. Also, specific for factor VIIa, which serinproteaza may occur fragmentation due to autocatalysis (enzymatic cleavage). Thus, the safety and effectiveness of any protein composition is directly related to its resistance. Maintaining stability in liquid form, as a rule, differs from liofilizovannyh form because of significantly increased the movements of the molecules and, consequently, to an increased likelihood of molecular interactions. Maintaining stability in concentrated form is also different because of the tendency to form aggregates at high concentrations of protein.

When developing liquid compositions take into account many factors. Kratkofil is fair, i.e. less than six months, the stability of the liquid, as a rule, depends on how it is possible to avoid explicit structural changes such as denaturation and aggregation. Such methods are described in the literature for many proteins, and there are many examples of stabilizers. It is well known that the agent is effective as a stabilizer for a single protein, actually acts as a destablizer for another. Once the protein is stabilized against explicit structural changes, development of liquid compositions with long term stability (i.e. more than six months) depends on further stabilization of the protein from the types of fracture, specific for this protein. More specific types of fracture can be, for example, the alignment of the disulfide bond, oxidation of some residues, the cyclization. Although certain types of destruction is not always possible to specify precisely developed tests to monitor subtle changes in order to control the possibility of specific excipients to stabilize exclusively protein of interest.

In addition to considerations of sustainability, as a rule, choose excipients, endorsed by various world medical control authorities. It is desirable that the pH of the composition after injection/infusion were in a physiologically suitable intervals the Le, otherwise, the result for the patient may be pain and discomfort.

For a General idea of protein compositions, see, for example, Cleland et al., The development of stable protein compositions: A closer look at protein aggregation, deamidation and oxidation, Critical Reviews in Therapeutic Drug Carrier Systems, 1993, 10(4):307-377; and Wang et al., Parenteral compositions of proteins and peptides: Stability and stabilizers, " Journal of Parenteral Science and Technology, 1988 (Supplement), 42(25).

Other publications dealing with interest in the stabilization of proteins, are listed below.

In U.S. patent 20010031721 A1 (American Home Products) refers to a highly concentrated, liofilizovane and liquid compositions of factor IX.

In U.S. patent 5770700 (Genetics Institute) relates to liquid compositions of factor IX.

WO 97/19687 (American Red Cross) relates to liquid compositions of plasma proteins, including factor VIII and factor IX.

In U.S. patent 4297344 describe the stabilization of coagulation factors II and VIII, anti-thrombin III and plasminogen against the action of heat by adding selected amino acids such as glycine, alanine, hydroxyproline, glutamine, and amino acid and carbohydrate such as a monosaccharide, oligosaccharide or sugar alcohol.

Factor VIIa undergoes a cascade of some reactions of destruction, in particular aggregation (dimerization), oxidation and autocatalytic cleavage (cleavage of the main chain peptide). In addition, there may be deposition. Many of these reactions which may greatly slow down the removal of water from the protein. However, the development of water composition for factor VIIa has the advantages of eliminating error recovery, and due to this increased precision, and ease of clinical use of the product, thereby increasing the flexibility of the patient's treatment. Ideally, the composition of factor VIIa should be stable for more than 6 months in a wide range of protein concentrations. This creates flexibility in the means of introduction. Generally, the more concentrated forms allow you to enter a smaller volume, which is highly desirable from the point of view of patients. Liquid compositions can have a number of advantages over the products dried by freezing, from the viewpoint of ease of introduction and application.

Currently, the only commercially available composition obtained by recombinant method FVII polypeptide is dried by freezing the product with factor FVIIa, which restore before applying; it contains relatively low concentrations of factor VIIa, for example, about 0.6 mg/ml Vial (1.2 mg) NovoSevenâ(Novo Nordisk, A/S, Denmark) contains 1.2 mg recombinants human factor VIIa, of 5.84 mg NaCl, 2,94 mg CaCl2.2H2Oh, of 2.64 mg GlyGly, 0.14 mg of Polysorbate 80 and 60.0 mg mannitol; restore it to pH 5.5 2.0 ml of water for injection (WFI). When recovering the protein solution Usto is sustainable for use within 24 hours. Thus, to date there are no commercially available ready for use liquid or concentrated products with factor VII.

Accordingly, in this area there is a need to improve the stability of the factor VII polypeptides, including human factor VIIa (chemical and/or physical resistance), increased concentration, maintaining activity levels and provide liquid compositions suitable for storage. Thus, the purpose of this invention is an aqueous composition of a polypeptide factor VIIa, which provides a reasonable regulation of the products of chemical and/or physical destruction, such as the products of the enzymatic cleavage or autocatalysis.

The invention

The present invention shows that the factor VII or its analogs (“factor VII polypeptide”), entered into the composition together with a buffer agent and a salt of calcium or magnesium or mixtures thereof in a concentration of at least 15 mm, stable in the range of pH from about 4 to about 8.

In one aspect the present invention relates to a liquid aqueous composition comprising (i) a factor VII polypeptide, (ii) a substance that is suitable for keeping pH in the range of from about 4 to about 8, and (iii) a substance selected from a list that includes calcium salt, magnesium salt sludge is a mixture, where the concentration of (iii) is at least 15 mm.

In various embodiments, the substance (iii) is present in a concentration of at least about 25 mm, 50 mm, 100 mm, 200 mm, 400 mm, 800 mm, 900 mm, or at least 1000 mm.

In another embodiment, the composition also contains (iv) the ionic strength modifier.

In various embodiments, the ionic strength modifier (modifier), which are selected from a neutral salt such as sodium chloride, amino acids or short peptides, or a mixture of at least two of these modifiers. In the preferred embodiment the ionic strength modifier is sodium chloride.

In various embodiments, the substance (iv) is present in a concentration of at least about 5 mm, 10 mm, 20 mm, 50 mm, 100 mm, 200 mm, 400 mm, 800 mm, 1000 mm, 1200 mm, 1500 mm, 1800 mm, 2000 mm or at least 2200 mm.

In one series of embodiments, the substance (iii) (calcium and/or magnesium salt) is present in a concentration of from about 15 mm to about 1000 mm, for example from about 25 mm to about 1000 mm, from about 50 mm to about 1000 mm, from about 100 mm to about 1000 mm, from about 200 mm to about 1000 mm, from about 300 mm to about 1000 mm, from about 400 mm to about 1000 mm, from about 500 mm to about 1000 mm, from about 600 mm to about 1000 mm from about 700 mm to about 1000 mm; from about 15 mm to about 800 mm, from about 25 mm to about 800 mm, from primer is 50 mm to about 800 mm, from about 100 mm to about 800 mm, from about 200 mm to about 800 mm, from about 300 mm to about 800 mm, from about 400 mm to about 800 mm, from about 500 mm to about 800 mm, from about 15 mm to about 600 mm, from about 25 mm to about 600 mm, from about 50 mm to about 600 mm, from about 100 mm to about 600 mm, from about 200 mm to about 600 mm, from about 300 mm to about 600 mm; about 15 mm to about 400 mm, from about 25 mm to about 400 mm, from about 50 mm to about 400 mm, or from about 100 mm to about 400 mm.

In one series of embodiments, the substance (iv) (modifier ionic strength) is present in a concentration from about 5 mm to about 2200 mm, for example from about 25 mm to about 2200 mm, from about 50 mm to about 2200 mm, from about 100 mm to about 2200 mm, from about 200 mm to about 2200 mm, from about 400 mm to about 2200 mm, from about 600 mm to about 2200 mm, from about 800 mm to about 2200 mm, from about 1000 mm to about 2200 mm, from about 1200 mm to about 2200 mm, from about 1400 mm to about 2200 mm, from about 1600 mm to about 2200 mm, from about 1800 mm to about 2200 mm, or from about 2000 mm to about 2200 mm; from about 5 mm to about 1800 mm, from about 25 mm to about 1800 mm, from about 50 mm to about 1800 mm, from about 100 mm to about 1800 mm, from about 200 mm to about 1800 mm, from about 400 mm to approx the RNO 1800 mm, from about 600 mm to about 1800 mm, from about 800 mm to about 1800 mm, from about 1000 mm to about 1800 mm, from about 1200 mm to about 1800 mm, from about 1400 mm to about 1800 mm, from about 1600 mm to about 1800 mm; from about 5 mm to about 1500 mm, from about 25 mm to about 1400 mm, from about 50 mm to about 1500 mm, from about 100 mm to about 1500 mm, from about 200 mm to about 1500 mm, from about 400 mm to about 1500 mm, from about 600 mm to about 1500 mm, from about 800 mm to about 1500 mm, from about 1000 mm to about 1500 mm, from about 1200 mm to about 1500 mm; from about 5 mm to about 1200 mm, from about 25 mm to about 1200 mm, from about 50 mm to about 1200 mm, from about 100 mm to about 1200 mm, from about 200 mm to about 1200 mm, from about 400 mm to about 1200 mm, from about 600 mm to 1200 mm, or from about 800 mm to about 1200 mm.

In one preferred embodiment the total concentration of substances (iii) and (iv) is from about 50 mm to about 2500 mm, for example from about 100 mm to about 2500 mm, from about 200 mm to about 2500 mm, from about 400 mm to about 2500 mm, from about 600 mm to about 2500 mm, from about 800 mm to about 2500 mm, from about 1000 mm to about 2500 mm, from about 1200 mm to about 2500 mm, from about 1400 mm to about 2500 mm, from about 1600 mm to about 2500 mm, from about 1800 mm to the ome 2500 mm, or from about 2000 mm to 2500 mm; from about 50 mm to about 2000 mm, from about 100 mm to about 2000 mm, from about 200 mm to about 2000 mm, from about 400 mm to about 2000 mm, from about 600 mm to about 2000 mm, from about 800 mm to about 2000 mm, from about 1000 mm to about 2000 mm, from about 1200 mm to about 2000 mm, from about 1400 mm to about 2000 mm, or from about 1600 mm to about 2000 mm; from about 50 mm to about 1600 mm, from about 100 mm to about 1600 mm, from about 200 mm to about 1600 mm, from about 400 mm to about 1600 mm, from about 600 mm to about 1600 mm, from about 800 mm to about 1600 mm, from about 1000 mm to about 1600 mm, or from about 1200 mm to about 1600 mm.

In one embodiment the substance (iii) and (iv) are present in concentrations of from about 600 mm to about 800 mm (iii) and from about 0 mm to about 5 mm (iv); in another embodiment the substance (iii) and (iv) are present in concentrations of from about 300 mm to about 500 mm (iii) and from about 1100 mm to about 1300 mm (iv); in another embodiment the substance (iii) and (iv) are present in concentrations of from about 100 mm to about 300 mm (iii and from about 1500 mm to about 1900 mm (iv); and in another embodiment the substance (iii) and (iv) are present in concentrations from about 50 mm to about 150 mm (iii) and from about 1800 mm to about 2300 mm (iv).

In various embodiments the ionic strength of the composition is at least about 50, NAP is emer, at least 75, 100, 150, 200, 250, 400, 500, 650, 800, 1000, 1200, 1600, 2000, 2400, 2800 or at least 3200.

In various embodiments, the calcium salt is selected from calcium chloride, calcium acetate, calcium gluconate and lunulata calcium. In various embodiments of the magnesium salt is selected from magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate, lunulata magnesium and salts of strong acids.

In preferred embodiments, the substance (iii) is selected from the list, which includes calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate or a mixture thereof; and an ionic strength modifier (iv) is sodium chloride.

In another embodiment, the composition also contains (v) modifier toychest.

In various embodiments, the modifier toychest (v) choose from the neutral salts, mono-, di - or polysaccharide, a sugar alcohol, amino acid or short peptide or a mixture of at least two of these modifiers.

In one embodiment, the modifier toychest (v) is present in a concentration of from about 1 to about 500 mm, from about 1 to about 300 mm, from about 10 to about 200 mm, or from about 20 to about 150 mm.

In another embodiment, the composition also contains (vi) non-ionic surface-active agent.

In one embodiment the nonionic surfactant is present in an amount of from about 0.005-note the RNO 2.0 wt.%.

In various embodiments the nonionic surfactant is a Polysorbate or poloxamer or simple alkyl ester of polyoxyethylene, preferably, poloxamer 188 or poloxamer 407, or Polysorbate 20 or Polysorbate 80, or poliakrilovye ether 23.

In another embodiment, the composition also contains (vii) an antioxidant. In various embodiments, the antioxidant is a D - or L-methionine; the analogue of methionine; methioninamide peptide; ascorbic acid; cysteine; homolog of methionine, such as homocysteine; glutathione. In the preferred embodiment the antioxidant is a L-methionine. In one embodiment the antioxidant is present in a concentration of from about 0.1 to about 5.0 mg/ml

In one embodiment the pH of the composition is maintained at a level of from about 4.0 to about 7.0 and, for example, from about 4.5 to about 7,0, from about 5.0 to about 7.0 and, from about 5.5 to about 7.0 and or from about 6.0 to about 7,0.

In one embodiment the substance is suitable for keeping pH in the range of from about 4.0 to about 8.0 a, is a buffering agent selected from the list consisting of acids and salts: citrate, acetate, histidine, malate, phosphate, tartaric acid, succinic acid, MES, HEPES, imidazole, Tris, lactates, glycylglycine, PIPES, glycine or a mixture of at least two of these buffer agents.

what one embodiment, the concentration of the buffer agent is from about 1 mm to about 100 mm, from about 1 mm to about 50 mm, from about 1 mm to about 25 mm, from about 2 mm to about 20 mm or about 10 mm.

In another embodiment, the composition also contains (viii) a preservative. In one embodiment, the preservative is selected from a list that includes phenol, benzyl alcohol, orthocresol, metacresol, paracresol, methylparaben, propylparaben, benzalkonium chloride and chloride benzathine.

In one embodiment the composition is isotonic, in another she is hypertensive. In one embodiment the composition is for pharmaceutical administration. In one embodiment, the composition is stable and/or stable for at least 6 months at 2-8aboutC.

In various embodiments the factor VII polypeptide is human factor VIIa; recombinant human factor VIIa; a polypeptide, a related factor VII; variant sequence of factor VII or a factor VII polypeptide, where the activity of the factor VII polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least 1.25 times, preferably at least about 2.0 or about 4.0, most preferably at least about 8.0 a, test “Analysis of in vitro proteolysis”, described in the present description. In one embodiment the factor VII polypeptide has a glycosylation that is different about the human factor VII wild-type.

In various embodiments the factor VII polypeptide is present in a concentration from about 0.1 mg/ml to about 10 mg/ml, from about 0.5 mg/ml to about 5.0 mg/ml, from about 0.6 mg/ml to about 4.0 mg/ml, from about 1 mg/ml to about 4.0 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.0 mg/ml, from about 0.1 mg/ml to about 2 mg/ml or from about 0.1 mg/ml to about 1.5 mg/ml

In another aspect, the invention also relates to a method of producing a liquid aqueous composition of a factor VII polypeptide comprising the stage of providing the factor VII polypeptide in a solution containing (i) a factor VII polypeptide; (ii) a substance that is suitable for keeping pH in the range of from about 4.0 to about 8.0 a; (iii) a substance selected from a list that includes a calcium salt, a magnesium salt or a mixture thereof; where the concentration of (iii) is at least 15 mm.

In another aspect, the invention also relates to the application of the composition to obtain a therapeutic agent for the treatment of syndrome, sensitive to factor VII.

In another aspect, the present invention relates to a method of treating syndrome, sensitive to factor VII, including an introduction to the subject in need of it, under conditions that result in less bleeding and/or increased blood clotting, an effective amount of aqueous liquid to which notizie, containing (i) a factor VII polypeptide, (ii) a substance that is suitable for keeping pH in the range of from about 4.0 to about 8.0 a, (iii) a substance selected from a list that includes a calcium salt, a magnesium salt or a mixture thereof; where the concentration of (iii) is at least 15 mm.

In various embodiments syndrome selected from the group consisting of hemophilia a, hemophilia b, deficiency factor XI deficiency factor VII, thrombocytopenia, von Willebrand disease, the presence of an inhibitor of coagulation factors, operations, intracerebral hemorrhage, trauma and anticoagulation therapy.

The compositions of the present invention is suitable as a sustainable and preferably ready-to-use compositions of factor VII polypeptides. The compositions are stable for at least six months, and preferably 36 months when stored at temperatures ranging from 2 to 8°C. the Composition is stable chemically and/or physically, when stored for at least six months at 2-8°C.

It is understood that “sustainable” means that the composition after storage for 6 months at 2-8°C retains at least 50% of its initial biological activity when measured in a one-step analysis of coagulation, in essence, that which is described in WO 92/15686 (example II). Briefly, the test obrzezameblowe in 50 mm Tris (pH 7.5), 0,1% BSA, and 100 μl is incubated with 100 μl of plasma with deficiency of factor VII and 200 μl of thromboplastin C containing 10 mm CA2+. Measure the clotting time and compared with a standard curve using an internal standard or a pool of normal human plasma with the addition of citrate in serial dilution.

Preferably, a stable composition retains at least 80% of its initial activity after storage for 6 months at 2-8°C.

It is implied that the term “stable”, which can be used interchangeably with the term “relatively stable”means that the composition after storage for at least 6 months at 2-8°C contains fewer at least one of the following products of destruction: (i) products of enzymatic degradation, (ii) aggregates (dimers, oligomers, polymers), (iii) oxidized forms, (iv) delivrande forms, relative to the number of the corresponding(s) of product(s) destruction, contained in the solution recovered product NovoSeven®which was kept in similar conditions during the same time period.

The term “physically stable” is supposed to denote a composition which remains visually transparent. Physical stability of the compositions evaluated by visually the test after storage of the compositions at different temperatures for different periods of time. Visual inspection of the compositions is carried out in a sharply focused light beam with a dark background. The composition is classified as physically unstable when it shows a visible haze.

The term “physical stability” of factor VII polypeptides refers to the formation of insoluble and/or soluble aggregates in the form of a dimeric, oligomeric and polymeric forms of factor VII polypeptides, as well as any structural deformation and denaturation of the molecule.

The term “clinically stable” is supposed to denote a composition which retains at least 50% of its initial biological activity after storage for 6 months at 2-8aboutWhen the measurement in single-stage analysis to collapse, essentially, that which is described in WO 92/15686.

It is implied that the term “chemical stability” refers to the formation of any chemical changes in the factor VII polypeptides after storage in solution under conditions of rapid processes. Examples are hydrolysis, deliciousa and oxidation, and enzymatic cleavage, leading to the formation of fragments of factor VII polypeptides, in particular, structuralia amino acids are prone to oxidation with the formation of the corresponding sulfoxidov.

Compositions containing the polypeptides of the factor VI, calcium ions and/or magnesium, buffer agents and, optionally, other excipients, which also stabilize the factor VII polypeptides, including ionic strength modifiers and modifiers toychest. The concentration of factor VII polypeptide ranges from about 0.1 to about 10 mg/ml

Used in this description, the term “ionic strength modifier” refers to substances that contribute to the ionic strength of the solution. Such substances are neutral salts, such as sodium chloride or potassium chloride; amino acids, short peptides (for example, 2-5 amino acid residues, such as, for example, glycylglycine) or a mixture of at least two of these modifiers and other substances. The preferred substance is sodium chloride. The ionic strength modifiers are present in a concentration of at least about 5 mm, 10 mm, 20 mm, 50 mm, 100 mm, 200 mm, 400 mm, 800 mm, 1000 mm, 1200 mm, 1500 mm, 1800 mm, 2000 mm or at least 2200 mm.

Used in this description, the term “modifier toychest” refers to substances that contribute to osmollnosti solution. Modifiers of toychest are amino acids, short peptides (for example, 2-5 amino acid residues), a neutral salt, mono - or disaccharides, polysaccharides, sugar alcohols, or a mixture of at least two of these modifiers and others in the society. Examples of modifiers toychest are sodium chloride, potassium chloride, sodium citrate, sucrose, glucose, glycylglycine and mannitol and other substances. Typically, the modifiers are present in a concentration of from about 1 to about 500 mm, from about 1 to about 300 mm, from about 10 to about 200 mm, or from about 20 to about 150 mm, depending on the other ingredients present. You can use a neutral salt such as sodium chloride or potassium chloride.

“Neutral salt” is a salt which, when dissolved in aqueous solution is neither acid nor base.

The term “substance, suitable for keeping pH in the range of from about 4.0 to about 8.0 a” includes those substances which maintain the solution pH in an acceptable range from about 4.0 to about 8.0 a, for example from about 4.0 to about 7.0 and, from about 4.5 to about 7,0, from about 5.0 to about 7.0 and from about 5.0 to 6.5, from about 5.5 to about 7.0 and, from about 5.5 to 6.5, from about 6.0 to about 7.0 and from about 5.0 to about 6.0 and from about 6.4 to about 6.6, or from about 5.2 to about 5,7 or about 5.5. The term can be used interchangeably with the term “buffering agent”. Such substances can be acid and salts: citrate (sodium or potassium), acetates (ammonium, sodium or calcium), Geest the Dean (L-histidine), malate, phosphate (sodium or potassium), tartaric acid, succinic acid, MES, HEPES, imidazole, Tris, lactates, glutamate, glycylglycine, PIPES, glycine, or a mixture of at least two of these buffer agents and other substances. The range of concentration of the buffer is chosen to maintain the preferred pH of the solution. A buffering agent may also be a mixture of at least two buffer agents, where the mixture is able to provide a pH value in a particular interval. In other embodiments, the buffer concentration is in the range from about 1 mm to 100 mm, from 1 mm to about 50 mm, from about 1 mm to about 25 mm, from about 2 mm to about 20 mm or equal to about 10 mm.

Optionally, the composition may also contain a surfactant or detergent. For “surface active substances” or “detergent”as a rule, are substances that protect the protein from stresses arising at the interface of air/solution, and stresses at the interface solution/surface (for example, leading to aggregation of the protein). Detergent, preferably, is a non-ionic detergent, which include Polysorbate (for example, twin®), such as Polysorbate 20 or 80, simple alkylether of polyoxyethylene or poloxamer, such as poloxamer 188 or 407 (for example, the polyols pluronic®), and others which affected the copolymers of ethylene/propylene, or polyethylene glycol (PEG), such as PEG8000, and other substances. The number present surfactant ranges from roughly 0.005 to about 2.0 percent.

Optionally, the composition may contain an antioxidant. The antioxidants include ascorbic acid, cysteine, homocysteine, cystine, cystathionine, methionine, glutathione and other peptides containing cysteine or methionine, in particular peptides with 2-5 amino acid residues, where at least one of the residues is a residue of methionine or cysteine, and other substances; it is preferable methionine, in particular L-methionine. The antioxidant include in a concentration of 0.1-5 mg/ml, for example 0,1-4, 0,1-3, 0,1-2 or 0.5-2 mg/ml

The composition can also include a preservative to delay microbial growth and thereby there is a possibility of packing factor VII polypeptides “reuse”. The preservatives include phenol, benzyl alcohol, orthocresol, metacresol, paracresol, methylparaben, propylparaben, benzalkonium chloride and chloride benzathine. Preservatives, typically include a concentration of 0.1-20 mg/ml, depending on the range of pH and type of preservative. Optionally, the composition may also contain a substance capable of inhibiting deliciousa.

In this description of a specific number should be same as the value in p is adalah ±10%, for example, about 50 mm assumes 50 mm ± 5 mm; for example, 4% assumes 4% ± 0,4%, etc.

Percentages are mass (mass/mass) as in the case when they refer to solids dissolved in the solution, and the liquids mixed in solutions. For example, for a twin it is the ratio of the weight 100% original solution/mass of solution.

The term “ionic strength” represents the ionic strength of the solution (µ), which is determined by the equation m =1/2S([I](Zi2)), where µ represents the ionic strength, [I] represents the molar concentration of the ion, and Z1represents the charge (+ or -) ion (James Fritz and George Schenk: Quantitative Analytical Chemistry, 1979). In various embodiments of the invention the ionic strength of the composition is at least 50, for example at least 75, 100, 150, 200, 250, 400, 500, 650, 800, 1000, 1200, 1600, 2000, 2400, 2800 or at least 3200.

The term “isotonic” means “isotonic with serum, i.e. approximately 300 ± 50 milliosmoles/kg. Toychest is intended to measure the osmolality of the solution prior to introduction. The term “hypertension” is intended to denote levels of osmolality above the physiological level of serum, for example, above 300 ± 50 milliosmoles/kg

The term “pharmaceutically effective amount” or “effective amount” reflects the effective dose, which the op is etelaat a qualified Clinician, which can offroute dosage to achieve the desired reaction. When determining the dose takes into account such factors as power, bioavailability, desirable pharmacokinetic/pharmacodynamic profiles, conditions of treatment, factors associated with the patient (e.g., weight, health, age etc), have jointly introduced drugs (e.g., anticoagulants), time of introduction or other factors known to medical practice.

The term “treatment” is defined as using subject and care about him, such as a mammal, including man, for the purpose of combating the disease, condition or disorder, and includes the introduction of the factor VII polypeptide to prevent the onset of symptoms or complications, or alleviating the symptoms or complications, or eliminating the disease, condition or disorder. The pharmaceutical compositions of the present invention, containing the factor VII polypeptide, you can enter parenteral subjects in need of such treatment. Parenteral administration can be accomplished by subcutaneous, intramuscular or intravenous injection with a syringe, optional syringe, reminiscent of the handle. On the other hand, parenteral administration can be accomplished using an infusion pump.

The concentration of factor VIIa is usually expressed in mg/ml or IU/ml, and 1 mg usually is 43000-56000 IU or more.

Applications

The preparations of the present invention can be used to treat any of the syndromes that are sensitive to factor VII, such as, for example, with bleeding disorders, including, without limitation, disorders caused by deficiency of coagulation factors (e.g., hemophilia a and b or deficiency of coagulation factors XI or VII); by thrombocytopenia or von Willebrand's disease, or inhibitors of coagulation factors, or excessive bleeding for any reason. Drugs can also be entered patients in connection with surgery or other trauma, or patients receiving anticoagulant therapy.

The factor VII polypeptides in the compositions according to the present invention

The terms “human factor VII” or “FVII” refers to human factor VII, obtained by different methods, including extraction from a natural source and purification, and use of recombinant systems for culturing cells. His consistency and characteristics are specified, for example, in U.S. patent No. 4784950. The terms also cover biologically active equivalents of the human factor VII, for example, distinguished by one or more amino acids in all sequences. In addition, terms used in this application are intended to include variants of factor VI with replacement, the deletion and insertion of amino acids or posttranslational modifications. Used in this description, the term “factor VII polypeptide” encompasses, without limitation, factor VII, as well as polypeptides, related to factor VII. For polypeptides, related to factor VII include, without limitation, factor VII polypeptides that are chemically modified relative to human factor VII and/or contain one or more changes in amino acid sequence relative to human factor VII (i.e. variants of factor VII), and/or contain truncated amino acid sequences relative to human factor VII (i.e. fragments of factor VII). Such polypeptides, related to factor VII may be different relative to human factor VII properties, including stability, binding of phospholipids, altered specific activity, etc.

It is implied that the term “factor VII” encompasses polypeptides of factor VII in their unsplit (imagenow) form, as well as polypeptides processed proteoliticeski with the formation of their respective bioactive forms, which can be called the factor VIIa. Typically, the factor VII is cleaved between residues 152 and 153 with the formation of factor VIIa. This also implies that the term “factor VII” encompasses, without limitation, polypeptides with aminokislotnoi sequence 1-406 of human factor VII wild-type (described in U.S. patent No. 4784950), as well as factor VII wild-type, derived from other species, such as, for example, factor VII, cows, pigs, dogs, mice, and salmon. It also includes natural allelic variation of factor VII that may exist and occur from one and another individual. The degree and localization of glycosylation or other posttranslational modifications may vary depending on the chosen host cells and the nature of the host cell environment.

Used in this description, the term “polypeptides, related to factor VII” encompasses, without limitation, polypeptides exhibiting essentially the same or improved biological activity relative to human factor VII wild type. Such polypeptides include, without limitation, factor VII or factor VIIa, chemically modified, and variants of factor VII, which introduced specific changes in amino acid sequence, modifying or destroying the biological activity of the polypeptide.

The term also encompasses polypeptides with a slightly modified amino acid sequence, for example polypeptides with modified N-end, including deletions or additions of N-terminal amino acids and/or polypeptides chemically modified relative factor VIIa person.

Polypeptides, odstvennye factor VII, including variants of factor VII, are essentially the same or improved biological activity relative factor VII wild type include, without limitation, polypeptides with the amino acid sequence differing from the sequence of factor VII wild type due to insertion, deletion or substitution of one or several amino acids.

Polypeptides, related to factor VII, including variants, having essentially the same or improved biological activity relative factor VII wild-type cover polypeptides exhibiting activity constituting at least about 25%, preferably at least about 50%, preferably at least about 75%, preferably at least about 100%, preferably at least about 110%, preferably at least about 120%, and most preferably at least about 130%of the specific activity of factor VIIa wild-type received in the cellular system of the same type when tested in one or more analyses from the analysis coagulating activity, the analysis of proteolysis or analysis of TF binding described in this description.

In some embodiments the factor VII polypeptides are polypeptides, related to factor VII, in particular variants where the ratio of AK is Yunosti specified factor VII polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least approximately 1.25 when tested, described in “Analysis of in vitro hydrolysis (see below, section “Tests”); in other embodiments the ratio is at least approximately 2,0; in other embodiments, the ratio is at least about 4.0. In some embodiments of the invention, the factor VII polypeptides are polypeptides, related to factor VII, in particular variants where the ratio of the activity of the specified factor VII polypeptide and the activity of native factor VIIa person (wild-type FVIIa) is at least approximately 1.25 when tested as described in the “Analysis of in vitro proteolysis” (see below, section “Tests”); in other embodiments the ratio is at least approximately 2,0; in other embodiments, the ratio is at least about 4.0; in other embodiments, the ratio is at least about 8,0.

In some embodiments the factor VII polypeptide is human factor VII, described, for example, in U.S. patent No. 4784950 (factor VII (wild-type). In some embodiments the factor VII polypeptide is human factor VIIa. In one series of embodiments the factor VII polypeptide is a polypeptide exhibiting activity constituting at least about 90%, preferably at least about 100%, preferably at least about 120%, predpochtitelnei - at least about 140%, most preferably at least about 160%, from the specific activity of human factor VIIa.

In some embodiments the factor VII polypeptides have an amino acid sequence differing from the sequence of factor VII wild type due to insertion, deletion or substitution of one or more amino acids.

In one series of embodiments the factor VII polypeptides include polypeptide exhibiting at least about 70%, preferably at least about 80%, preferably at least about 90%, most preferably at least about 95%, identity with the sequence of factor VII wild type disclosed in U.S. patent No. 4784950. Homology/identity to the amino acid sequence is usually determined from the rows of sequences using a suitable computer program for the ranked sequence, such as, for example, the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22:4673-4680).

Non-limiting examples of factor VII, which has essentially the same or improved biological activity compared with factor VII wild type, include S52A-FVII, S60A-FVII (during closing et al., Arch. Biochem. Biophys., 352:182-192, 1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K37A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII and S336G-FVII; FVIIa variants exhibiting increased TF-independent activity described in WO 01/83725 and WO 02/22776; FVIIa variants exhibiting increased proteolytic stability as described in U.S. patent No. 5580560; factor VIIa, split proteoliticeski between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng., 48:501-505, 1995); and oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys., 363:43-54, 1999).

The biological activity of factor VII polypeptides

The biological activity of factor VIIa in the blood clotting process comes from its ability (i) to contact with tissue factor (TF) and (ii) to catalyze the proteolytic cleavage of factor IX or factor X with the formation of activated factor IX or X (factor IXa or XA, respectively).

For the purposes of the invention, the biological activity of factor VII polypeptides (“biological activity of factor VII”) can be quantified by measuring the ability of the drug promote the clotting of blood using a plasma deficiency of factor VII and thromboplastin, as described, for example, in U.S. patent No. 5997864 or in WO 92/15686. If the above analysis, the biological activity is expressed as the decrease in clotting time relative to con the full sample and converted into units of factor VII by comparing with the standard mixed human serum, containing 1 unit/ml activity of factor VII. On the other hand, the biological activity of factor VII may be quantified,

- measuring the ability of factor VIIa or a polypeptide related to factor VII, to produce activated factor X (factor XA) in the system containing TF, enclosed in a lipid membrane and factor X. (Persson et al., J. Biol. Chem., 272:19919-19924, 1997);

- measuring the degree of hydrolysis of factor X in the aqueous system (“Analysis of in vitro proteolysis”, see below);

- measuring the physical binding of factor VIIa or polypeptide, a related factor VIIa, TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts, 413:359-363, 1997); and

- measuring the degree of hydrolysis of a synthetic substrate by factor VIIa and/or polypeptide, a related factor VIIa (“Analysis of in vitro hydrolysis”, see below); and

- measuring the thrombin generation in vitro TF-independent system.

Assays suitable for determining the biological activity of factor VII polypeptides

The factor VII polypeptides useful in accordance with the present invention, it is possible to choose with the help of suitable assays that can be implemented as a simple preliminary tests in vitro. Thus, in the present description discloses a simple test (called “Analysis of in vitro hydrolysis”) on the activity of factor VII polypeptides.

And the Alize in vitro hydrolysis (analysis 1)

Native (wild-type) factor VIIa and factor VII polypeptide (hereinafter both referred to as “factor VIIa”) can be analyzed for specific activity. They can also be analyzed in parallel to directly compare their specific activities. The analysis is carried out in titrations the microplate (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mm, is added to the factor VIIa (final concentration 100 nm) in 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 5 mm CaCl2and 1 mg/ml bovine serum albumin. The absorption at 405 nm was measured continuously in the apparatus for reading tablets SpectraMax™ 340 (Molecular Devices, USA). Absorption, developing during the 20-minute incubation, after subtraction of the absorbance in control wells containing no enzyme, is used to calculate the relationship between the activities of the polypeptide of factor VII and factor VIIa wild-type.

Ratio = (A nm of factor VII polypeptide)/(A nm factor VIIa wild type).

On the basis of obtained results it is possible to identify the factor VII polypeptides with an activity of less comparable or higher than that of the native factor VIIa, such as, for example, the factor VII polypeptides, where the ratio of the activity of the factor VII polypeptide to the activity of native factor VII (FVII wild type) is about, or the example is about 1,0.

The activity of factor VII polypeptides also can be measured using a physiological substrate, such as factor X (“Analysis of in vitro proteolysis”), at a suitable concentration range of 100 to 1000 nm, where the measure generated factor XA after addition of a suitable chromogenic substrate (e.g., S-2765). In addition, analysis of the activity can be carried out at physiological temperature.

Analysis of in vitro proteolysis (analysis 2)

Native (wild-type) factor VIIa and factor VII polypeptide (hereinafter both referred to as “factor VIIa”) analyze in parallel to directly compare their specific activities. The analysis is carried out in titrations the microplate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nm) and factor X (0.8 μm) in 100 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 5 mm CaCl2and 1 mg/ml bovine serum albumin, incubated for 15 minutes Then cleavage of factor X is stopped by adding 50 μl of 50 mm Hepes, pH 7.4, containing 0.1 M NaCl, 20 mm EDTA and 1 mg/ml bovine serum albumin. The number of obrazovavshegosya factor XA is measured by adding a chromogenic substrate Z-D-Arg-Gly-Pro-p-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mm. The absorption at 405 nm was measured continuously in the apparatus for reading tablets SpectraMax™ 340 (Molecular Devices, USA). Absorption, developing for 10 minutes, after subtraction of the absorption in the completed hole, not containing FVIIa, is used for calculating the ratio between the proteolytic activities of the polypeptide of factor VII and factor VIIa wild-type.

Ratio = (A nm of factor VII polypeptide)/(A nm factor VIIa wild type).

On the basis of obtained results it is possible to identify the factor VII polypeptide with the activity of smaller, comparable, or higher, than those of the native factor VIIa, such as, for example, the factor VII polypeptide, where the ratio of the activity of the factor VII polypeptide to the activity of native factor VII (FVII wild type) is about or about 1.0.

The ability of factor VIIa or factor VII polypeptides to generate thrombin can be measured in the analysis (analysis 4), containing all the relevant coagulation factors and inhibitors at physiological concentrations (net of factor VII, when simulated condition of hemophilia a) and activated platelets (as described on p.543 in the work of Monroe et al. (1997), Brit. J. Haematol., 99, 542-547, included in this description by reference).

The activity of factor VII polypeptides also can be measured using a single-stage analysis on coagulation (analysis 4), in essence, therefore, as described in WO 92/15686 or in U.S. patent 5997864. Briefly, the test sample is diluted in 50 mm Tris (pH 7.5), 0.1% of BSA, and 100 μl is incubated with 100 μl of plasma with deficiency of factor VII and 200 μl of trombla is Tina, containing 10 mm CA2+. Measure the clotting time and compared with a standard curve using an internal standard or a pool of normal human plasma with the addition of citrate in serial dilution.

Production and purification of factor VII polypeptides

Purified human factor VII, suitable for use in the present invention, preferably, produced using recombinant DNA technology, for example, as described in Hagen et al., Proc. Natl. Acad. Sci. USA, 83:2412-2416, 1986, or as described in European patent No. 200421 (ZymoGenetics, Inc.). Factor VII can also be obtained by the methods described in Broze and Majerus, J. Biol. Chem., 255(4):1242-1247, 1980, and Hedner and Kisiel, J. Clin. Invest., 71:1836-1841, 1983. These methods give the factor VII without detectable amounts of other blood clotting factors. You can get even more purified preparation of factor VII to include the quality of the final stage of purification of additional gel filtration. Then factor VII in turn activated form VIIa by known methods, for example, using several different plasma proteins, such as factor XIIa, IX or CA. On the other hand, as described in Bjoern et al. (Research Disclosure, 269, September, 1986, pp. 564-565), factor VII can be activated by passing it through a column for ion exchange chromatography, such as Mono Q®(Pharmacia fine Chemicals) or the like, or by autoactivation in which aStore.

Polypeptides, related to factor VII can be obtained by modification factor VII wild-type or recombinant technology. Polypeptides, related to factor VII, modified, compared with factor VII wild-type amino acid sequence can be obtained by modification of the nucleotide sequence encoding the factor VII wild-type, or change the amino acid codons or by removal of some amino acid codons in the nucleic acid that encodes a natural factor VII, known methods such as site-specific mutagenesis.

For specialists in the art it will be obvious that you can replace the outside of the regions critical to the function of a molecule of factor VIIa, even leading to active polypeptide. Amino acid residues essential for the activity of the factor VII polypeptide, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science, 244:1081-1085). In the latter method, the mutation is introduced into every positively charged residue in the molecule, and the molecule-mutants have coagulation activity, respectively, the activity of cross-stitching, to identify amino acid mod is s, which are critical for activity of the molecule. Place of interaction of substrate-enzyme can also be determined by analysis of three-dimensional structures when determining by methods such as nuclear magnetic resonance, crystallography or photoaffinity tagging (see, for example, de Vos et al., 1992, Science, 255:306-312; Smith et al., 1992, Journal of Molecular Biology, 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

Introduction mutations in nucleating sequence for the substitution of one nucleotide for another nucleotide can be site-directed mutagenesis using any method known in the art. Particularly suitable is a procedure that uses a vector super helical conformation was double-stranded DNA insert of interest and two synthetic primers containing the desired mutation. Oligonucleotide primers, each complementary to opposite chain vector, complete it during the temperature cycle using DNA polymerase Pfu. After the introduction of the primers generates a mutated plasmid containing staggered nicks. Upon completion of the temperature cycle, the product is treated with Dpnl specific for methylated and paleometeorology DNA for cleavage of the parental DNA template and to select the synthesized DNA containing the mutation. It is also possible to use other known in the art procedures for POPs the project, identification and selection of options, such as, for example, methods of permutation of genes and phage reflection.

The selection of polypeptides from the cell from which they originated, can be achieved by any method known in the art, including without limitation, removing the cell culture medium containing the desired product of the fused cell culture, centrifugation, or filtration to remove Eclipsys cells; and the like.

Optionally, the factor VII polypeptides can be cleaned separately. Cleaning can be accomplished using any method known in the art, including, without limitation, affinity chromatography, such as, for example, on a column with antibodies against factor VII (see, for example, Wakabayashi et al., J. Biol. Chem., 261:11097, 1986; and Thim et al., Biochem., 27:7785, 1988); hydrophobic chromatography; ion exchange chromatography; gel-chromatography; electrophoretically procedures (e.g., preparative isoelectric focusing (IEF)), differential dissolution (e.g., ammonium sulfate precipitation), or extraction, and similar methods. See, for an overall view, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After cleaning, the preparation preferably contains less than about 10 wt.%, preferably less than about 5%, most preferably less than the approximately 1%, factors, which is not a factor VII polypeptides derived from the host cell.

The factor VII polypeptides can be activated by proteolytic cleavage, using factor XIIa or other proteases with trypsin-like specificity, such as, for example, factor IXa, kallickrein, factor XA and thrombin. See, for example, Osterud et al., Biochem., 11:2853 (1972); Thomas, U.S. patent No. 4456591; Hedner et al., J. Clin. Invest., 71:1836 (1983). On the other hand, the factor VII polypeptides can be activated by passing them through a column for ion exchange chromatography, such as Mono Q®(Pharmacia), or similar column. Then the activated factor VII polypeptide can be incorporated into the composition and type, as described in this application.

Description of figures

Figure 1 shows the content of the units FVII and fragments of FVII after 3 months of storage at 2-8aboutC.

The following examples illustrate the practical application of the invention. These examples are only for purposes of illustration and are in no way intended to limit the scope of the invention.

Experimental examples

Example 1

Methods of analysis

The content of the units is determined not causing denaturation gel-chromatography - HPLC. The content of oxidized forms define RP-HPLC. The content of the forms, formed by enzymatic cleavage, Oprah is elaut RP-HPLC.

Do not cause denaturation gel chromatography carried out on a column of Waters Protein Pak 300 SW, 7.5 x 300 mm, using as mobile phase 0.2 M solution of ammonium sulfate, 5% 2-propanol, pH 7.0. A flow rate of 0.5 ml/min. and Detection at 215 nm. Download FVIIa 25 mcg.

HPLC with reversed phase carried out on a column (4.5 mm x 250 mm with patented silicon dioxide with attached butyl rubber particle size of 5 μm and pore size E. The column temperature 70aboutC. Buffer A: 0,1% (vol./about.) triperoxonane acid. Buffer: 0,09% (vol./about.) triperoxonane acid, 80% (vol./about.) acetonitrile. Column elute with a linear gradient from X to (X+13)% In 30 minutes. X select so that eluted FVIIa with a retention time of approximately 26 minutes. The flow rate of 1.0 ml/min. and Detection at 214 nm. Download FVIIa 25 mcg.

Example 2

Getting songs

In General, samples of water compositions FVIIa for analysis in these experimental examples, which are obtained from purified primary solution by exchanging the buffer to the column for gel filtration. Additives for compositions or contained in the buffer for elution in their final ratios or added to the eluate. The resulting solution is sterilized by filtration using a sterilized membrane filter (pore size 0.2 μm or equivalent) and poured into sterile glass LAF is ony, stoppered and sealed with butyl rubber stoppers and aluminum caps, which sergivita.

Example 3

The effect of pH on the chemical/physical stability

The vials of water composition rFVIIa containing 1.4 mg rFVIIa/ml, 50 mm sodium chloride, 10 mm calcium chloride and a mixture of 10 mm glycylglycine, acetate and histidine, in which the pH is brought to 3, 3,5, 4,0, 4,5, 5,0, 5,5, 6,0, 6,5, 7,0, 7,5, 8,0, 8,5 and 9.0, or incubated at a temperature of 2-8aboutWith or at elevated storage temperature - 30aboutWith, then extract at different points in time and analyze the content to changes in pH, determine the chemical stability of the method RP-HPLC and the total HPLC.

After storage at 2-8aboutIn the period up to three months water compositions show little change in pH. Does not cause denaturation of gel-chromatography - HPLC, performed on samples stored for up to three months at 2-8aboutWith, does not show significant aggregation drugs at values of pH = 5,5 (figure 1). RP-HPLC performed on the same samples show a significant increase fragmentation or oxidation of protein in the range of pH 4.5-5.5.

Figure 1 shows the results after 3 months of storage at 2-8aboutC. the Initial content of aggregates is approximately 0.5%, and the initial content of the fragments is PR is approximately 9%.

Example 4

The buffer capacity of the various buffers

The vials of water composition rFVIIa containing 1.0 mg rFVIIa/ml, 50 mm sodium chloride, 10 mm calcium chloride in a concentration of 10 mm in one of the buffer substances from among glycylglycine, malic acid, acetic acid, histidine, glutamic acid and citric acid, or incubated at a temperature of 2-8aboutWith, or at high temperatures - 30aboutWith in 3 months. At time zero the pH was adjusted to 5.5, because at this pH have the least amount of decay products (figure 1). The pH of the composition containing glycylglycine, shows his rise to 6.2 during storage. Other songs in the same period show a stable value of 5.5±0,1.

Example 5

Physical stability of aqueous compositions containing various detergents

Get twelve different compositions. The composition includes

rFVII - 0.75 mg/ml,

NaCl of 2.92 mg/ml,

CaCl2.2H2O to 1.47 mg/ml,

glycylglycine - 1,32 mg/ml,

detergent/solubilizer - x mg/ml;

pH 5.5.

The concentration of the test detergents/solubilization listed in the table below.

The composition is obtained from the liquid core solution rFVII. The starting solutions of detergents/solubilization receive buffers containing NaCl, CaCl2.2H2O and gli is iglitzin, in the concentrations mentioned above. The main solution of rFVIIa and the solutions of the detergent is mixed and the pH of the solutions was adjusted to 5.5. The composition was filtered (0.2 μm) and filled into vials (1 ml per vial).

The appearance of the compositions determined by visual inspection and determine the absorption of the composition at 400 nm. Then the vials shaken for 19 hours (800 rpm) at room temperature. After shaking define the appearance and the absorption at 400 nm. The results are shown below in the table.

The type of detergentConc. (mg/ml)AppearanceAbsorbance (400 nm)
ToAfterToAfterIncrease increase
Without (standard)-little frequent.Very muddy0,00851,43861,4301
Twin®800,1souse is little frequent. Transparent, little frequent.0,00440,00360,0008
Twin®200,1very little frequent.Transparent, little frequent.0,00390,01010,0062
Poloxamer 1881,0very little frequent.Transparent, little frequent.0,00630,00270,0036
Pluronic®F1271,0very little frequent.Transparent, little frequent.0,00000,00480,0048
The polyethylene glycol 4000,1very little frequent.Muddy0,00761,57081,5632
Polyethylene glycol 40000,5the Alo frequent. Very muddy0,01081,66241,6516
Brij®350,1very little frequent.Transparent, little frequent.0,00280,00150,0013
Myrj®590,1very little frequent.Transparent, little frequent.is 0.00020,11100,1108
Myrj®520,1very little frequent.Transparent, little frequent.0,00090,93900,9381
LPCM0,1very little frequent.Transparent, little frequent.0,00260,0012-0,0014
Glycerin1,0very little frequent. Muddy0,00401,40641,4024

“frequent.” = “particles”

The results show that the pattern (without adding any detergent/solubilizer) becomes visually cloudy when shaken, and there was a significant increase in absorption at 400 nm. Adding twin®20 (= Polysorbate 20), twin®80 (= Polysorbate 80), poloxamer 188, pluronic®F127 (= poloxamer 407), Brij®35 (= poliakrilovye ether 23) and LPCM (= myristoyl-α-lysophosphatidylcholine) almost completely prevents the increase in turbidity and absorption, while in the case of Myrj®59 (= polyxystra 100) and Myrj®52 (= polyxystra 40) compared to the benchmark see a smaller increase in turbidity. Glycerin, polyethylene glycol 400 or polyethylene glycol 4000 not prevent an increase in turbidity in concentrations used in this experiment.

Example 6

Chemical stability of aqueous compositions containing methionine as an antioxidant

Get three different compositions. The compositions contain

rFVIIa - 0.75 mg/ml,

NaCl of 2.92 mg/ml,

CaCl2.2H2O to 1.47 mg/ml,

glycylglycine - 1,32 mg/ml,

methionine is 0 or 0.25 or 1.0 mg/ml;

pH 6.5.

The composition is obtained from the liquid core solution of rFVIIa. Methionine dissolved in buffers containing NaCl, CaCl2.2H2O and glycylglycine concentrations specified above. The main solution of rFVIIa and solutions of methionine are mixed and the pH of the solutions was adjusted to 6.5. The composition was filtered (0.2 μm) and filled into vials (1 ml per vial). Vials stored at 5°C, 25°C and 40°C. Samples of extract and analyze the content of the oxidized forms (RP-HPLC) at the time specified in the table below. The table shows the content of oxidized forms.

td align="justify"> 1,9
Methionine (mg/ml)Time zero25°C, 14 days40°C, 14 days25°C, 28 days40°C, 28 days5°S, 90 days
0 (standard)2,44,47,54,412,83,1
0,251,72,45,32,89,9
1,01,62,35,02,69,61,3

The results show that the addition of methionine reduces the degree of oxidation in the composition.

Example 7

Chemical stability of aqueous compositions containing calcium chloride

Get four different compositions. The compositions contain

rFVIIa - 1.0 mg/ml,

NaCl of 2.92 mg/ml,

CaCl2.2H2O to 1.47 mg/ml (10 mm)of 29.4 mg/ml (200 mm), 58,8

mg/ml (400 mm) and of 117.6 mg/ml (800 mm)

accordingly,

glycylglycine - 1,32 mg/ml,

pH 7.0.

The composition is obtained from the liquid core solution of rFVIIa. Calcium chloride is dissolved in buffers containing NaCl and glycylglycine, and after mixing with the main solution receive rFVIIa concentrations mentioned above. After mixing, the pH of the solutions was adjusted to 7.0. The composition was filtered (0.2 μm) and filled into vials (1 ml per vial). Vials stored at 5aboutC.

The activity of factor VII (IU/ml) determined by analysis of collapse.

The content of calcium chloride in the composition0 mdr, IU/ml3 mdr, IU/ml, 5about
1 (10 mm)5444433623
2 (200 mm)5991746528
3 (400 mm)5468059370
4 (800 mm)5177352801

1. Liquid aqueous composition containing
(i) a factor VII polypeptide;
(ii) a substance that is suitable for keeping pH in the range of from about 4 to about 8;
(iii) a substance selected from calcium salts, magnesium salts or mixtures thereof,
where the concentration of (iii) is at least 15 mm,
and (vii) an antioxidant.

2. The composition according to claim 1, additionally containing (iv) the ionic strength modifier.

3. The composition according to claim 2, where the ionic strength modifier (iv) are selected from a neutral salt such as sodium chloride, amino acids or short peptides, or a mixture of at least two of these modifiers.

4. The composition according to claim 3, where the ionic strength modifier (iv) is sodium chloride.

5. Composition according to any one of claims 1 to 4, where the compound (iii) is present in a concentration of at least about 25 mm, such as at least about 50 mm, 100 mm, 200 mm, 400 mm, or at least 800 mm.

6. The composition according to claim 2 where the compound (iv) is present in Konz is Tracii at least about 5 mm, for example at least about 10 mm, 20 mm, 50 mm, 100 mm, 200 mm, 400 mm, 800 mm, 1000 mm, 1200 mm, 1500 mm, 1800 mm, 2000 mm or at least 2200 mm.

7. The composition according to claim 1, where a calcium salt selected from calcium chloride, calcium acetate, calcium gluconate and lunulata calcium.

8. The composition according to claim 1, where the magnesium salt is selected from magnesium chloride, magnesium acetate, magnesium sulfate, magnesium gluconate and lunulata magnesium.

9. Composition according to any one of PP 8, wherein the ingredient (iii) is selected from calcium chloride, calcium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, or mixtures thereof; and where the ionic strength modifier (iv) is sodium chloride.

10. The composition according to claim 1, additionally containing (v) a substance modifying toychest.

11. The composition of claim 10, where the substance modifying toychest (v), choose from the neutral salts, mono-, di - or polysaccharide, a sugar alcohol, amino acid or short peptide, or a mixture of at least two of these modifiers.

12. The composition of claim 10 or 11, where the substance modifying toychest (v), is present in a concentration from about 1 mm to about 500 mm.

13. The composition according to item 12, where the concentration is 10-250 mm.

14. The composition according to claim 1, additionally containing (vi) non-ionic surface-active agent.

15. The composition according to 14, where the nonionic surfactant is from the battle Polysorbate, or poloxamer, or simple alkyl ester of polyoxyethylene, for example, poloxamer 188, poloxamer 407, Polysorbate 20, Polysorbate 80 or poliakrilovye ether 23.

16. The composition according to claim 1, where the antioxidant (vii) are selected from L - or D-methionine analogue of methionine, methioninamide peptide, ascorbic acid, cysteine, homocysteine, glutathione, cysteine, and cystathionine.

17. The composition according to item 16, where the antioxidant is an L-methionine.

18. Composition according to any one of p-17, where the antioxidant is present in a concentration of from about 0.1 to about 5.0 mg/ml, for example from about 0.1 to about 4 mg/ml, from about 0.1 to about 3 mg/ml, from about 0.1 to about 2 mg/ml, or from about 0.5 to about 2 mg/ml

19. The composition according to claim 1, where the pH is maintained at a level of from about 4.0 to about 7.0 and, for example, from about 4.5 to about 7,0, from about 5.0 to about 7.0 and, from about 5.5 to about 7.0 and or from about 6.0 to about 7,0.

20. The composition according to claim 1, where the substance is suitable for keeping pH in the range of from about 4.0 to about 7.0 and choose from acids and salts: citrates, acetates, histidinol, malatov, phosphate, tartaric acid, succinic acid, MES, HEPES, imidazole, Tris, lactates, glycylglycine, PIPES, glycine, or a mixture of at least two of these substances.

21. The composition according to claim 20, where the concentration is from prima is but 1 mm to about 50 mm.

22. The composition according to item 21, where the concentration of the buffer is about 10 mm.

23. The composition according to claim 1, additionally containing (viii) a preservative such as phenol, benzyl alcohol, ortho-cresol, meta-cresol, para-cresol, methylparaben, propylparaben, benzalkonium chloride or chloride benzathine.

24. The composition according to claim 1, which is isotonic.

25. The composition according to claim 1, formulated for pharmaceutical administration.

26. The composition according to claim 1, which is stable for at least 6 months at 2-8°C.

27. The composition according to claim 1, where the factor VII polypeptide is human factor VIIa, preferably human factor VIIa, obtained by recombinant methods.

28. The composition according to claim 1, where the factor VII polypeptide is a variant of the sequence of factor VII.

29. The composition according to p, where the ratio of the activity of the factor VII polypeptide to the activity of native human factor VIIa (wild-type FVIIa) is at least 1.25 times, preferably at least about 2.0 or about 4.0, most preferably at least about 8.0 a, test "Analysis of in vitro proteolysis", described in the present description.

30. The composition according to claim 1, where the factor VII polypeptide is present in a concentration from about 0.1 mg/ml to about 10 mg/ml, for example from about 0.5 mg/ml to about 5.0 mg/ml, from about 0.6 mg/is l to about 4.0 mg/ml, or from about 1.0 mg/ml to about 4.0 mg/ml

31. The method of producing a liquid aqueous composition of a factor VII polypeptide comprising the stage of providing the factor VII polypeptide in a solution containing (ii) a substance that is suitable for keeping pH in the range of from about 4.0 to about 8.0 a, (iii) a substance selected from calcium salts, magnesium salts or mixtures thereof, where the concentration of (iii) is at least 15 mm and an antioxidant.

32. The use of a composition according to any one of claims 1 to 30 for receiving a therapeutic agent for the treatment of syndromes that are sensitive to factor VII.

33. A method of treating syndrome, sensitive to factor VII, including an introduction to the subject in need, an effective amount of aqueous liquid composition containing (i) a factor VII polypeptide, (ii) a substance that is suitable for keeping pH in the range of from about 4.0 to about 8.0 a, (iii) a substance selected from a list that includes a calcium salt, a magnesium salt or a mixture where the concentration of (iii) is at least 15 mm.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to operative gynecology and concerns prevention of complications in hysteromyoma patient in case of hysterectomy. For this purpose during 14 days before and 14 days after operation mediation selmevit is introduced perorally. Dose is 1 tablet a day.

EFFECT: performing supravaginal amputation or uterus extirpation against the background of chronic posthemorrhagic anemia, reduction of intraoperative hemorrhage and prevention of DIC syndrome development.

3 tbl, 2 ex

FIELD: chemistry; pharmacy.

SUBSTANCE: invention claims novel [1,2,4]triazolo[1,5-a]pyrimidine-2-ylurea derivatives of the general formula (1), or their pharmaceutically acceptable salts: [formula 1] , where Ar is optionally substituted phenyl group, dihydrobenzofuranyl or thiophenyl group. X is O. R is group of formula (2)-(6) or , where values of Cy and R4-R12 radicals are given in the claim, and medicine containing these compounds.

EFFECT: obtaining compounds with immunosuppressive or immune tolerance inducing effect, which can be applied in medicine.

14 cl, 16 tbl, 333 ex

FIELD: medicine.

SUBSTANCE: described NASP may be introduced as independent agents or in combination with other medications (such as factors VIII and VIIIa) to provide hemostasis. Method is also described for treatment of individual, who needs improvement of coagulation, which includes introduction of therapeutically efficient amount of composition containing non-anticoagulating sulphated polysaccharide (NASP) to specified individual.

EFFECT: wide range of procoagulant agent application.

19 cl, 8 dwg, 13 tbl, 7 ex

FIELD: medicine; pharmacology.

SUBSTANCE: offered are compositions of formula 1 , where PG represents hydrogen or formyl group. R1 and R2 together with nitrogen atom to which they are attached, form heterocycle chosen from piperidine or morpholine and their pharmaceutically acceptable salts.

EFFECT: high haemostatic activity.

8 ex

FIELD: medicine.

SUBSTANCE: haemostatic compressing bandage represents appliance, having guide rail with fixed on it one-piece spiked clamping plates (one of them is still, another is moving) and rotating drum with ratchet gear. Between the still plate and cylinder drum, which is fixed on guide rail, armoured belt made of non-hygroscopic air-penetrable material is set with movable hygroscopic swab/bolster. The haemostatic compressing bandage is impregnated with new original haemostatic medicinal composition, including ingredients as follows (by weight): N-(β-oxymethyl)-4,6-dimethyl-dihydropirimidone-2 (xymedone) - 20.0-22.0%, phenylephrine (mesatone) - 2.0-2.5%, decilate - 1.0-1.2%, boric acid - 9.5-10.0%, aqueous 50% glycerol solution.

EFFECT: increase in efficiency of external hemorrhage arresting.

3 cl, 7 dwg

FIELD: preparative biochemistry, proteins.

SUBSTANCE: invention relates to a method for preparing albumin-enriched fraction with the decreased content of prekallikrein activator (PKA) and albumin-containing fraction with the reduced content of prekallikrein activator (PKA). Invention proposes a method for preparing albumin-enriched plasma fraction with the decreased content of prekallikrein activator (PKA) that involves the following steps: (a) reducing paste V prepared by fractionation according to Cohn's method by suspending paste V at temperature 2 ± 2°C for 6 h in 1.6-fold water mass for injection at pH = 7.2-7.6; (b) step for concentrating fraction prepared at step (a); (c) heating fraction prepared at step (b) in the range of temperature 50-70°C for time providing pasteurization of fraction; (d) package of the prepared fraction for using, and (e) carrying out the incubation step under the following conditions: 10 days at temperature 30-32°C or 4 weeks at 20-25°C. Invention provides decreasing content of PKA in fractions prepared from plasma containing albumin, and preparing albumin-containing fraction with the decreased content of PKA.

EFFECT: improved preparing method.

5 cl, 3 tbl, 3 ex

FIELD: pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to preparing medicinal formulations as film membranes in aims for providing hemostatic, wound-healing, anti-inflammatory effect in medicinal practice. Membrane comprises furacillin as a medicinal component, chitosan as a film-forming agent and dimethylsulfoxide, aerosil, 98% acetic acid for preparing its 2% solution and water as accessory substances. The novel medicinal membrane possesses the prolonged curative effect. Invention expands region for using the membrane.

EFFECT: valuable medicinal properties of membrane.

7 tbl, 3 dwg, 1 ex

FIELD: medicine, experimental medicine.

SUBSTANCE: for the purpose to stop hemorrhages it is necessary to introduce fibrin-monomer for a rabbit at the dosages ranged 0.5-5 mg/kg animal body weight. In case of intravenous injection at the dosages ranged 0.5-5 mg/kg animal body weight fibrin-monomer is of immediate hemostatic action and has no influence upon basic hemostatic parameters.

EFFECT: higher efficiency.

5 ex

FIELD: medicine, hematology, polypeptides.

SUBSTANCE: invention discloses compositions comprising factor VII or polypeptide relates to factor VII, and PAI-1 or PAI-1-related polypeptide, and for using this composition in treatment of bleeding. Invention discloses a method for treatment of bleeding in a patient, a method for decreasing the coagulation time in a patient, a method for enhancing hemostasis in a patient, a method for increasing the fibrinolysis time in a patient, a method for enhancing clots strength in a subject, a set designated for treatment of bleeding attack. Invention provides the development of compositions that can be used effectively in treatment or prophylaxis of bleeding in blood coagulation disorders, in particularly, the development of compositions as a single, standard medicinal formulation that can be used effectively in treatment or prophylaxis of bleeding or as a procoagulant, the development of compositions, methods for treatment and sets using of that promotes to displaying the synergetic effect, the development of compositions, methods for treatment and sets without essential adverse effects, for example, high level of systemic activation of the blood coagulation system.

EFFECT: improved and valuable medicinal properties of polypeptides and pharmaceutical composition.

53 cl, 4 dwg

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to haemostatic glue powder. Proposed powder represents a mixture containing sodium alginate and feracryl taken in the weight ratio = 1:(1-3). Using a powder provides ceasing bleeding and after contacting with blood and tissues it forms a film for about 10-30 s. Also, powder doesn't stick to hands.

EFFECT: improved and valuable medicinal properties of powder.

6 ex

FIELD: medicine.

SUBSTANCE: present invention refers to medical products, particularly to large depletion mixture containing the following components per litre of aqueous solution: polyethylene glycol 90 to 150; ascorbic acid and/or ascorbic acid salt 5 to 15; alkaline or earth metal sulphate or mixed alkaline or earth metal sulphates 5 to 10; and electrolyte (sodium chloride, potassium chloride and sodium hydrocarbonate). The mixture agents are chosen so that the osmolarity of the aqueous solution reduced to 1 litre is within 300 to 550 mOsmoles/litre. Besides the invention concerns a cleaning agent, a component set for large intestine depletion, to application of polyethylene glycol as a large intestine depletion agent and to method of large intestine depletion.

EFFECT: higher mixture safety and tolerance.

39 cl, 29 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: present invention pertains to crystalline substance for peroral solid medicinal preparation, which is an indoline compound (KMD-3213), which exhibits blocking action to α1-adrinaline receptors, is suitable for use as a therapeutic medium in case of dysuria and is represented by formula (I) . The x-ray diffraction picture of the powder of this compound is characterised by main peaks 5.5°±0.2°, 6.1°±0.2°, 9.8°±0.2°, 11.1°±0.2°, 12.2°±0.2°, 16.4°±0.2°, 19.7°±0.2° and 20.0°±0.2°, as 2θ.

EFFECT: obtaining solid medicinal preparations for treating dysuria, containing this crystalline substance as an active ingredient.

14 cl, 3 dwg, 2 tbl, 9 ex

FIELD: medicine; pharmacology.

SUBSTANCE: composition contains, essentially anhydrous ordered (adhesive) mixture of, at least, one pharmaceutically active agent in the form of microparticles linked to surface of carrier particles which are essentially greater than those of the active agent or agents, and, essentially are water insoluble or poor soluble, in a combination with the agent enabling bioadhesion and/mucoadhesion, linked to surface of specified carrier particles. The composition, mainly, is used for sublingual or intranasal introduction. Besides, the invention refers to method of composition preparation.

EFFECT: improved bioadhesive properties, fast release of active substance.

31 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention includes as active substances one or several fibrates and one or several statins or their pharmaceutically comprehensible salt, in carrier chosen from group, consisting of i) admixtures of polyethyleneglycol and poloxamer in the ratio from 2:1 to 3:1 and with the subsequent dispersion on lactose, ii) glyceryl monostearate with the subsequent spraying on lactose or on an admixture of lactose and hydroxypropymethylcellulose; and iii) polyethyleneglycol, with subsequent spraying on Aeroperl. Besides, the invention concerns firm dosed out forms including specified material and way of their obtaining.

EFFECT: possibility to establish suitable biological availability of both active ingredients at peroral insertion.

56 cl, 15 tbl, 15 ex

FIELD: medicine, pharmacology.

SUBSTANCE: immobilised antibiotic drug of common formula B-F-A, where B is base representing calcium phosphate, or hydroxyphosphate (hydrohyapatite), or their mix, F is -P-O- group, implanted on surface of B, A is antibiotic residual, containing non-protonated amino and/or alcohol groups; and method for immobilised antibiotic production, comprising calcium phosphate, or hydroxyphosphate, or their mix, treatment with binding agent, followed by interreaction with antibiotic, containing non-protonated amino and/or alcohol groups; the binding agent used is phosphorus pentachloride; the process being conducted in aprotic solvent.

EFFECT: prolongation of antibiotic washing-off from the surface, antibiotic release in original form, and absence of toxic products resulted from immobilised antibiotic hydrolytic degradation.

5 cl, 7 ex, 8 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: pharmaceutical composition in the form of the solid medicinal form contains desmopressin in amount of therapeutically active ingredient.

EFFECT: enlarged period of validity of the specified active ingredient in the specified medicinal form.

15 cl, 1 ex, 9 tbl, 6 dwg

FIELD: medicine; dermatology.

SUBSTANCE: spend mesotherapy of the stretch marks area with an admixture of preparations the tsel-T and kutis-kompozitum at 1:1. Carry out injections immediately in area of dermal defects on 0.1 ml of a solution through every 2-3 mm. Then perform phonophoresis of Lydasums in the stretch mark area within 10-15 minutes. Alternate the specified procedures in day with carrying out of applications of cosmetic clay on the stretch marks area within 30-40 minutes at temperature of 40-50°C.

EFFECT: method excludes possibility of occurrence of allergic reactions and is simple in execution.

2 ex, 4 cl

FIELD: medicine.

SUBSTANCE: invention refers to anti-infective materials (agents) and methods of production thereof, characterised by biocompatibility with animal or human body liquids and tissues. Invention concerns method of production of anti-infective agent, production of anti-infective agent implying modification of inorganic mineral with silico- and alumooxycompounds, namely, Na-shaped bentonite, inorganic metal salts in polar solvent, followed by bentonite keeping in salt solution, isolation of promodified bentonite from solution and drying at temperature not higher 100°C. Before being modified bentonite is enriched with Na+ ions through processing with 3-10% aqueous solution of chloride sodium with following washing and filtering of produced semiproduct thereafter modified by 10-20% inorganic metal salts solution represented with silver nitrate or copper sulphate. Modified bentonite is kept in specified salt solutions within 12-24 hour. Then promodified bentonite is cleared from sodium salts through washing and filtration. After being dried made agent is milled to particle dispersion 20-150 nm. Thus inorganic mineral are processed with specified solutions in ratio, weight fraction bentonite:solution, as 1: (10-40).

EFFECT: production of agent with higher anti-infective efficiency using simple and cheap methods and materials.

7 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention reveals how to procure polypeptide forms in a solid state, in which the polypeptide is stabilised against degradation under physiological conditions or conditions that go beyond the limits for a long period of time. Stabilised polypeptide particles, which contain a polypeptide chosen from a super family polypeptide, which activates adenylate cyclase of the hypophysis/glucagons, and a stabilising agent chosen from disaccharides and monosaccharides, which are stable at a chosen acidic pH value, in this case the particles are made from a water solution, which has a chosen acidic pH value and are stable at temperatures till 60°C for a period of at least two months. The solid polypeptide particles under this particular invention provide stabilisation of the polypeptide, allowing them to achieve 96% restoration of the stabilised polypeptide after storage.

EFFECT: development of an effective way of getting stabilised polypeptide form in a solid state.

11 cl, 7 ex, 9 dwg, 8 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to urology and concerns prevention of urethra iatrogenic infection caused by transurethral manipulations. For this purpose 5 minutes prior to urinary tracts manipulation gel is introduced to urethra that contains 2 g of 10 % lydocaine solution per 100 g of glycerine. Before introduction gel is ozonised to ozone concentration 1200 mkg/l. Method provides effective prevention of urethra iatrogenic infection at absence of lydocaine by-effects.

EFFECT: provided effective prevention of urethra iatrogenic infection at absence of lydocaine by-effects.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ceruloplasmin recovery, and can be used in making enzyme blood-plasma medicines. The disclosed method includes dissolution of feed stock in acetate buffer, enzyme sorption in an ion-exchange reactor with functional DEAE group, elution and following purification. Said ion-exchange reactor with functional DEAE group represents a sorbent on fixed styroldivinylbenzene matrix with co-graft DEAE group. And in purification, at first the dissolved enzyme is treated with a solvent-detergent mixture. Then the treated enzyme is immobilised on sulphopropylcation sorbent, washed in acetate buffer containing sodium chloride. The purified enzyme is eluted, while the feed stock is dissolved in acetate solution. The enzyme is sorbed in the ion-exchange reactor wherein as such sorbent on fixed styroldivinylbenzene matrix with co-graft DEAE group is applied. The enzyme is eluted and purified. In purification, the enzyme solution is treated with a solvent-detergent mixture, immobilised on sulphopropylcation sorbent, washed in acetate buffer containing sodium chloride. The purified enzyme is eluted.

EFFECT: method ensures virus safety of recovered medical product ceruloplasmin and allows improving end product yield.

9 cl, 2 ex

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