Modified erythropoietin with attached water-soluble long-chain molecule

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceutics and deals with a pharmaceutical composition, which contains feline erythropoietin as an active ingredient, to which two or more polyethyleneglycol molecules with a non-branched chain are attached, with a water-soluble long-chain molecule having the molecular weight, constituting not less than 30 kDa and producing the haemopoietic effect. A haemopoietic medication and a medication for the treatment of anaemia are based on the said composition.

EFFECT: group of inventions provides the haemopoietic effect, which lasts for not less than seven days, when introduced to humans and/or animals.

8 cl, 4 dwg, 2 tbl, 2 ex

 

Area of technology

The present invention relates to a pharmaceutical composition containing erythropoietin, which is attached to the water-soluble long-chain molecule.

Background of the invention

To alleviate anemia erythropoietin (sometimes using abbreviations EPO), a hormone which enhances hematopoiesis, produced using the methods of genetic recombination and used as a pharmaceutical product that provides good results in the treatment of anemia. It was found that the joining of polyethylene glycol (sometimes using the abbreviation "PEG"), which is a water-soluble long-chain molecule, to the EPO of human origin inhibits the metabolism of EPO in the liver, which lengthens the lifetime of EPO in the blood (patent document 1). Consequently, the effect of lengthening the period of time spent in the blood effect of the medicine lasts longer, reducing the frequency of administration. This advantage has attracted attention to medicines in the form of PEG-modified proteins and PEG-modified EPO forms as pharmaceutical products for the next generation. In fact, some of them have already been introduced into practical use.

When implementing modifications �tree using PEG protein is usually the harder it is metabolized the more the molecular weight of the attached PEG or the greater the number of attached PEG molecules. For this reason, expect the protein to which is attached a large number of high molecular weight PEG molecules, after in vivo administration, will have a longer duration of existence in the blood (non-patent document 1). On the other hand, in the case of proteins such as EPO, which exhibit physiological activity upon binding to a receptor, increasing the molecular weight of the attached water-soluble long-chain molecules and the number of attached molecules of physiological activity in vitro is reduced.

In the case when with EPO derived from human sequence (sometimes using abbreviations "EPO") was modified using PEG, PEG-modified EPO, in which one molecule of PEG attached to the lysine at position 52 (sometimes using abbreviations mono-Mahilyowskaya form") reportedly has excellent effect of renewal of hematopoietic activity (as indicated by the increase of reticulocytes) after administration in a single dose to rats via the tail vein (patent document 1).

With the modern use of medicines human EPO administered intravenously, subcutaneous, intramuscular and other ways. There had been further� attempts to develop, for example, orally and absorbed through the nasal mucosa drugs (patent document 2). Intravascular administration of drugs EPO person does not have wide applicability. Hence there is a need to preparations EPO, which is administered in an easier fashion, impose on the patient a small load, for example, cause less pain after the injection and, moreover, have long-term effectiveness of the drug.

The symptoms of anemia, reasons such as chronic renal failure, chemotherapy and surgery, which in accordance with the messages found in humans are also reported in domestic animals such as dogs and cats, and are often the causes of death of these animals. For example, in cats (in Japan) the incidence rate of chronic renal failure significantly increased from 0.64% to 3,95% over the ten year period beginning with 1996, up from 28th place to 4th place in the ranking of diseases (non-patent document 2). Drugs human EPO is therapeutically used for the treatment of anemia in the animal. However, since the physiologically active proteins such as EPO, have species-specific amino acid sequence, in the case of the introduction of non-human animal drugs EPO brow�ekeskog of origin there is a risk of the induction of the expression of antibodies against EPO. As a result at present, studies on the development of drugs EPO, each of which has an amino acid sequence specific for a particular species, for the treatment of cats or dogs.

Patent document 1: WO 02/32957

Patent document 2: JP-A S62-89627

Non-patent document 1: “Polyethylene glycol-conjugated pharmaceutical proteins”, EXPERTISE, Vol.1, № 8, 352-356 (1998).

Non-patent document 2: Tama Jūi Rinshō Kenkyūkai Chōsa Hōkoku (2006 Nendo) [2006 Report on Survey of Tama Veterinary Clinics Association].

A brief summary of the invention

The object of the present invention is to provide a pharmaceutical composition that contains EPO as an active ingredient, has a high level of safety in vivo, despite the presence of high physiological activity, and has the peculiarity consisting in the fact that after administration to a human and/or animal it causes hematopoietic effect that lasts for at least seven days.

The authors of the present invention have conducted thorough studies on the chemical modification of EPO using PEG, in which they found that depending on the number of attached PEG molecules and the molecular weight or structure of PEG hematopoietic effect lasts for at least seven days. The obtained results were the basis of the present invention.

Corre�idents, the present invention relates to pharmaceutical compositions comprising erythropoietin, which is attached two or more water-soluble long-chain molecules in which the number of erythropoietin is not less than 50% of the total content of erythropoietin.

The present invention furthermore relates to a pharmaceutical composition containing erythropoietin, which is attached to the water-soluble long-chain molecule, and a water-soluble long-chain molecule has a molecular weight equal to not less than 30 kDa.

The present invention, furthermore, relates to a pharmaceutical composition containing erythropoietin, which is attached to the water-soluble long-chain molecule, and a water-soluble long-chain molecule has a branched chain.

Preferably the water-soluble long-chain molecule represents at least one compound selected from the group consisting of polyethylene glycol, polyaminoacid and polypropylene glycol.

Pharmaceutical composition preferably has a hematopoietic effect when administered to a human and/or animal, which lasts for not less than seven days, and the animals are preferably animals of the family Felidae and/or the family Canidae.

Preferably the pharmaceutical�Kai composition is a solution or gel, a solution or gel with a pH of at least 4 but not more than 8.

Preferably the solution are such that it has an osmotic pressure and pH, essentially identical with those that have fluid in mammals, so as not to cause pain after injection.

Preferably erythropoietin has grown from human, cat or dog amino acid sequence.

Preferably erythropoietin is a polypeptide of (a) to(C):

(a) a polypeptide having the amino acid sequence defined in SEQ ID NO:1;

(b) a polypeptide which has an amino acid sequence identical to at least 70% amino acid sequence defined in SEQ ID NO:1 and has activity of erythropoietin in the analysis of cell proliferation using cells BaF/EPOR; or

(c) a polypeptide which has one or more substitutions, deletions, insertions and/or additions of amino acids relative to the amino acid sequence defined in SEQ ID NO:1 and has activity of erythropoietin in the analysis of cell proliferation using cells BaF/EPOR.

Preferably not less than one, the point of attachment of the polyethylene glycol is a residue of lysine-78.

Even after repeated introduction of the pharmaceutical composition preferably does not cause products ant�body against erythropoietin.

The present invention furthermore relates to a drug for the treatment of anemia and hematopoietic means, each of which includes the above pharmaceutical composition.

As a result of the introduction of pharmaceutical compositions of the present invention to a human or animal hematopoietic effect can last for at least seven days. Pharmaceutical composition of the present invention, in the case of drug administration, imposes on the patient a small load, for example, causes less pain after the injection and, moreover, has long-term effectiveness of the drug.

Brief description of the drawings

Fig.1 compares the hematopoietic effects caused by the introduction of the rat PEG-modified EPO cats, to which were added different amounts of PEG molecules with an unbranched chain, which has a molecular weight equal to 20000.

Fig.2 compares the hematopoietic effects caused by the introduction of the rat PEG-modified EPO cats, to which were added different amounts of PEG molecules with an unbranched chain, which has a molecular weight equal to 5000 (served as control mono-Paglierani EPO cats, to which was attached one molecule of PEG with an unbranched chain, which has a molecular weight, ravno� 20000).

Fig.3 compares the hematopoietic effects caused by the introduction of the rat PEG-modified EPO cats, to which was attached one or two PEG molecules with an unbranched chain, which has a molecular weight equal to 40000 (served as control mono-Paglierani EPO cats, to which was attached one molecule of PEG with an unbranched chain, which has a molecular weight equal to 20000).

Fig.4 compares the hematopoietic effects caused by the introduction of the rat PEG-modified EPO cats, to which was added one or two molecules of the branched PEG chain has a molecular weight equal to 20000 (served as control mono-Paglierani EPO cats, to which was attached one molecule of PEG with an unbranched chain, which has a molecular weight equal to 20000).

Best mode of carrying out the invention

1. The present invention relates to three pharmaceutical compositions.

The first aspect of the present invention relates to pharmaceutical compositions containing in an amount equal to not less than 50% of the total content of erythropoietin, erythropoietin (sometimes using abbreviations "EPO"), which is attached two or more water-soluble long-chain molecules.

In the first aspect of the present invention, the number of water-soluble donnazac�to link molecules attached to the EPO, is two or more, are preferred two molecules. Additionally, it may contain EPO, is attached to one water-soluble long-chain molecule. However, from the point of view of the duration of the hematopoietic effect of EPO, is attached to two or more water-soluble long-chain molecules, is preferably the main component, more preferably is at least 50% and even more preferably at least 70% of the total EPO content.

Here the percentage of EPO, is attached to two or more water-soluble long-chain molecules, based on the total content of EPO, is calculated by determining the fluorescence intensity of the bands obtained by staining with the use of fluorescent substances, which is followed by separation by electrophoresis in SDS-page.

What EPO, is attached to two or more water-soluble long-chain molecules, is a major component in the overall EPO, means that the EPO is attached to two or more water-soluble long-chain molecules, demonstrates the most intense band in SDS-page. Here the expression "demonstrates the most intense band in SDS-page" means that in the case of proteins contained in the sample subjected to electri�Reza in SDS-page, by staining with the use of fluorescent substances, using, for example, SYPRO Ruby or etc., the intensity of the signal from EPO, is attached to two or more water-soluble long-chain molecules, the higher the intensity of other signals.

To determine whether EPO, is attached to two or more water-soluble long-chain molecules, the main component in total EPO may also be used for HPLC. For example, the sample passing through the column, for example, YMC Pack Diol-200 (available in the market from YMC Co., Ltd.), detected by measuring the optical density, and can be determined that the signal intensity from EPO, is attached to two or more water-soluble long-chain molecules, the higher the intensity of other signals.

From the point of view of the duration of the hematopoietic effect of molecular weight water-soluble long-chain molecule is preferably at least 10 kDa and more preferably at least 20 kDa. When less than 5 kDa hematopoietic effect usually does not last long.

The second aspect of the present invention relates to pharmaceutical compositions containing EPO, to which is attached a water-soluble long-chain molecule, and a water-soluble long-chain molecule has a molecular weight of not less than 30 kDa.

In the second aspect� present invention, the number of water-soluble long-chain molecules attached to the EPO, and not subject to any particular limitation and may be equal to one molecule, or may be two or more molecules.

From the point of view of the duration of the hematopoietic effect of molecular weight water-soluble long-chain molecule is preferably at least 30 kDa and more preferably at least 40 kDa. When less than 20 kDa hematopoietic effect usually does not last long.

The third aspect of the present invention relates to pharmaceutical compositions containing EPO, to which is attached a water-soluble long-chain molecule, and a water-soluble long-chain molecule has a branched chain. The expression "water-soluble long-chain molecule has a branched chain, means that the number of branches is two or more, wherein the number of branch points is two or more.

In cases where water-soluble long-chain molecule is a branched chain, the number of water-soluble long-chain molecules that are attached to the EPO, and not subject to any particular limitation. Even in cases where the number of attached water-soluble long-chain molecules is equal to one molecule, the hematopoietic effects when administered pharmaceutical composition, as detected, for a period of not less than SEM� days. How to attach PEG having a branched chain described in JP-H9 T-504299.

Following are the water-soluble long-chain molecule, the method and the effect of administration, container and erythropoietin in the above pharmaceutical compositions.

2. Water-soluble long-chain molecule

Water-soluble long-chain molecule is attached to the EPO for the purpose of inhibiting the metabolism and improve the kinetics, characterized by the lengthening of time spent in the blood. Water-soluble long-chain molecule and not subject to any particular limitation, and examples include polyethylene glycol (PEG), polyaminoamide and polypropylene glycol. The use of such molecules may include preparation of an intermediate product, and then attach to the protein during the reaction synthesis. For the production of proteins that are associated with these molecules, can also be used methods of genetic recombination. Of the above molecules of PEG has no antigenicity and is non-toxic, and thus is also effective in reducing the antigenicity of the modified protein and the suppression of the expression of antibodies against EPO as a side effect.

Methods of covalent bonding of PEG to a protein, typically involve a chemical reaction with the protein or with activated oxidized lignite through extraction�Institute of functional group in its chain of sugars such as a polyol, lactol, amine, carboxylic acid or carboxylic acid derivative. There are also ways in which used activated with ether sulfonic polymer, such as activated with ether sulfonic PEG. In the case of accession to the EPO also may join by using such methods.

Intermediate reaction products of Paglierani that may be used include long chain molecules that were methoxylamine on one end. Furthermore, the development of intermediate products, which tarifitsirovana when using Succinimidyl-fatty acids on the other hematoxylineosin the end of the PEG; of these, those in which part in the form of fatty acid is propionic acid or butyric acid, are preferred from the viewpoint of reactivity. When educated Succinimidyl-propionic acid ester of methoxy-PEG (using the abbreviation "SPA-PEG") is reacted EPO human, accession, as it is known, occurs selectively to lysine residues. Since EPO has a lot of lysine residues, a reaction progress, the number of attached PEG molecules increases, leading to a mixture of isomers having different amounts of attached molecules.

It was reported that in the results, a semi�military after administration of a single dose of human EPO to rats via the tail vein, mono-Mahilyowskaya the form in which one molecule of PEG (20 kDa) is attached to lysine 52, produced the longest hematopoietic effect; however, the optimal ratio of isomers or of a mixture of components depends on the animal species from which the EPO, and the method of administration. And PEG-modified EPO in which the number of attached PEG molecules equal to one, and the PEG-modified EPO in which the number of attached PEG molecules is two or more, can be entered separately or you can enter in the mixture.

The point of attachment of PEG to protein EPO will likely depend on the type, from which, EPO, and type of reaction. However, even in cases of connection of a plurality of PEG molecules it is preferable that at least one point of joining was the lysine residue corresponding to lysine 52 in human EPO. For example, in EPO cats having the sequence defined in SEQ ID NO:1, it is preferred to attach to lysine 78.

3. Method and the effect of the introduction

Thus, EPO-containing pharmaceutical composition of the present invention produces a hematopoietic effect when administered to a human and/or animal, which lasts for not less than seven days.

As for hematopoietic effect, the ratio of reticulocytes (precursors of red blood cells) and red blood cells can be quantitatively op�idelity and use as an indicator of hematopoietic activity after administration of the pharmaceutical compositions of the present invention. The number of reticulocytes can be determined using a cervical smear using a colorant or dye, or by using an automatic counter of blood corpuscles.

The subject that is suitable for administration of pharmaceutical compositions, and not subject to any particular limitation; the introduction can be carried out individual and also animal than human. In non-human animals, particular restriction is imposed, although preferred are animals of the family Felidae and/or family Canidae. As will be described later, when using events from cats EPO for the animals of the family Felidae and the Canidae family, the probability that the EPO will act as an antigen, small, enabling to avoid occurrence of side effects.

Methods of administration of pharmaceutical compositions include, but without limitation, intravenous, subcutaneous, oral, intramuscular, transdermal and nasal administration.

PEG-modified EPO in the present invention can be used with a substance selected from pharmaceutically acceptable fillers that may cause disintegration agents, and binders.

Examples of fillers include starch, agar, sucrose, lactose, glucose, dextrin, sorbitol, acacia gum, corn starch, mannitol, crystalline powder package�Yu cellulose, lecithin, calcium phosphate and calcium sulfate. Can also be used pharmaceutically acceptable excipients that are different from these.

Examples causing disintegration agents include starch, agar, calcium citrate, calcium carbonate, sodium hydrogen carbonate, dextrin, crystalline cellulose, carboxymethylcellulose and tragakant. Can also be used pharmaceutically acceptable cause disintegration agents, different from these.

Examples of binders include starch and starch derivatives, cellulose and cellulose derivatives, acacia gum, tragakant, gelatin, sugar, ethanol and polyvinyl alcohol. Can also be used pharmaceutically acceptable binders which are different from these.

PEG-modified EPO in the present invention can be used with a substance selected from stabilizers, pH modifiers, osmotic pressure modifiers and surfactants.

Examples of stabilizers include amino acids. Here used as stabilizers amino acids can be in the form of crystals or may be amorphous. Alternative can be used amino acids, which include impurities such ingredients of plants or animals with a high degree of such amino acids. Used the crystals can be in the L-form, D-form or in kind� mixture of L - and D-forms.

Examples of pH modifiers include a buffer system selected from the group consisting of acetic acid/acetate, malic acid/malate, citric acid/citrate, tartaric acid/tartrate, lactic acid/lactate, phosphoric acid/phosphate, glycine/glycinate, Tris, glutamic acid/glutamate and sodium carbonate and other buffers. The lower limit of the pH of the gel or solution containing EPO, preferably equal to 4, more preferably 4.5 and even more preferably 5. The upper limit of the pH is preferably 8, more preferably 7.5 and even more preferably 7.

Examples of the osmotic pressure modifiers include, but are not limited to, salts, sugars, alcohols and amino acids. In particular, it is possible to use sodium chloride, polyhydric alcohols, Monohydric alcohols, monosaccharides, disaccharides, oligosaccharides and amino acids and their derivatives.

Examples of polyhydric alcohols that may be used include triatomic alcohols, such as glycerol, pentatomic alcohols, such as Arabic, xylitol and adonit, and setiathome alcohols, such as mannitol, sorbitol and dulcet. Of these, preferred are setiathome alcohols, and particularly suitable for use is mannitol.

Examples of Monohydric alcohols include methanol, ethanol and isopropyl alcohol. Of these, ethanol is�tsya preferred.

Examples of monosaccharides which may be used include sugars with five carbon atoms as a skeleton (a pentose such as arabinose, xylose, ribose and 2-deoxyribose, and sugar with six carbon atoms skeleton (hexose, such as glucose, fructose, galactose, mannose, sorbose, rhamnose and fucose. Of these, preferred are sugars consisting of six atoms of the carbon skeleton.

Examples of oligosaccharides that can be used include trisaccharide such as maltotriose and raffinoses, and tetrasaccharide, such as stachyose. Of these, preferred are trisaccharide.

Examples of derivatives of these monosaccharides, disaccharides and oligosaccharides, which can be used include glucosamine, galactosamine, gluconic acid and galacturonic acid.

In addition, a surfactant may be contained together with the PEG-modified EPO in the present invention. Given as examples of surfactants include anionic surfactants, nonionic surfactants, amphoteric surfactants and cationic surfactants, although not limited to these substances. Given as examples of anionic surfactants on�of despair, but without limitation, anionic surfactants based on fatty acids, anionic surfactant based on linear alkyl benzenes, anionic surfactants based on higher alcohols, anionic surface-active substances on the basis of α-olefins and anionic surfactants on the basis of normal paraffins. Given as examples of nonionic surfactants include, but are not limited to, nonionic surfactants based on fatty acids, nonionic surfactants based on higher alcohols and nonionic surfactants based on alkyl phenols. Illustrative non-limiting examples of nonionic surfactants include Polysorbate and/or alkyl esters polyoxyethylenesorbitan. Given as examples of amphoteric surfactants include, but are not limited to, amphoteric surfactants based on amino acids, betinov or aminoxide. Cited as examples of cationic surfactants include, but are not limited to, cationic surfactants based on Quaternary ammonium salts.

These above mentioned fillers, causing disintegration agents, binders, stable�congestion, modifiers of pH, osmotic pressure modifiers, and surfactants can be unlimitedly to combine. In addition, can be added pharmaceutically acceptable additives other than fillers, causing disintegration agents, binding agents, stabilizers, pH modifiers, osmotic pressure modifiers and surfactants. Their examples include lubricants, coating agents, colorants, dispersants, activators suction, soljubilizatory, materials for dietary products, food supplements, vitamins, flavoring substances, sweeteners, preservatives, preservatives and antioxidants.

To obtain pharmaceutical ingredients can be formulated in the form of a solution, gel or powder and subsequently transferred to the drug type of solution, lyophilized preparation, pre-filled syringe, the drug sustained-release subcutaneous implant, mizerny preparation, gel preparation or liposomal drug, for example.

When the EPO-containing pharmaceutical composition is used in the form of a solution, it is important to make a solution so that he had osmotic pressure and pH, similar to those of body fluids of mammals (osmolality: 280 mOsm/kg N2About; pH 7.4), Thu�would not cause pain during subcutaneous injection. When the EPO-containing pharmaceutical composition is used in the form of a solution, the composition having an osmotic pressure not to exceed 400 mOsm/kg N2Oh, can accordingly be used in the present invention. Can also be used accordingly compositions having an osmotic pressure of not less than 200 mOsm/kg N2O.

4. Container

Because EPO is effective in vivo even after the introduction of a small dose, losses due to adsorption of the drug to the wall of the container have a large impact on the actual activity after administration in vivo. Therefore, the container for this drug is preferably the polymer product, to which is adsorbed a small amount of protein. Accordingly, it is desirable to select the material for the container based on the conditions that the adsorption of EPO to the container does not exceed 1% of the total number of EPO in the container. It is preferable to select the material based on the condition that such adsorption preferably does not exceed 0.7% and more preferably of 0.5%.

For polymeric materials, which are known to be characterized by low adsorption of proteins, a special limitation is not imposed. Their examples include materials for containers for therapeutic applications, such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), �olycarbonate and polimetilmetakrilat. Preferred examples of polymeric materials that are known, are characterized by low adsorption of proteins include polymers, which are polymers obtained by polymerization with ring opening of cycloolefin, such as a norborene, tetracyclinea or its derivative, and hydrogenation products of these polymers; and copolymers, the molecular chain of which cyclopentenyl residue or substituted cyclopentadienyl residue incorporated during the polymerization of cycloolefin, such as a norborene, tetracyclinea or its derivative, with ethylene or propylene. Cycloolefin copolymers (COC), which are copolymers derived from tetrastromatica and the olefin, such as ethylene as starting materials are preferable because they have low adsorption. In addition, also preferred are cycloolefin polymers (COC), which are polymers obtained by polymerization with ring opening of norborene, and then hydrogenation (see JP-A H5-300939 or JP-A H5-317411).

5. Amino acid sequence of EPO

Amino acid sequence of the EPO may be a sequence derived from any organism. Was carried out the cloning of cDNA for EPO, for example, mice, rats, dogs and cats, as well as human�ka (Wen et al. Blood 82, 1507 (1993)), were clarified and amino acid sequences that encode these EPO. In this invention can be used not only human EPO, but also EPO having the amino acid sequence specific to a species different from man.

Preferably the amino acid sequence of the EPO in the present invention is the amino acid sequence originating from a man or amino acid sequence derived from an animal of a detachment of the Carnivora. In the unit Carnivora family Felidae and the Canidae family are preferred, with the family Felidae is preferred.

It is known that EPO cats have identical sequences of human EPO on to 83.4%. Also EPO cats, expressed in cells SNO, has hematopoietic activity in cats (Am. J. Vet. Res. 64, 1465-71 (2003)).

The causes of anemia, as it is known, include massive hemorrhage, vitamin deficiency, autoimmune diseases, malignant tumors, chronic inflammation, chronic renal failure, hemolysis and impaired hematopoiesis. Anemia caused by these reasons is observed not only in humans but also in domestic animals such as dogs and cats, livestock, such as cattle, horses, sheep, goats and pigs, and even animals such as lions, tigers, Kang�, elephants, giraffes, zebras, koalas and pandas, which are grown and publicly exhibited for environmental studies at the Zoological gardens, etc., Such cases of anemia can be treated by the EPO used in the present invention. EPO cats can be used to treat anemia in animals of the family Felidae, such as cats, lions and tigers. Furthermore, the EPO cats identical to a high degree of sequence EPO dogs and is also effective in dogs and other animals of the family Canidae, which taxonomically belongs to the order Carnivora.

Unlike humans, due to the lack of system reserve of preserved blood for transfusion above animals find it hard to take action in the form of infusions of blood, for example in the case of blood loss due to trauma or during surgery. However, such animals can be used, either instead of transfusion, or during drip infusion, the drug EPO of the present invention, which produces long-hematopoietic effect. If the animal feels pain during insertion, it may prevent the introduction of, and in the case of large animals may even be dangerous to the vet. However, the pain can be minimized by the installation of osmotic pressure and pH at values close to the values in the liquid in the organism of the animal.

Preferably� amino acid sequence of EPO, derived from animals of the family Felidae, is the amino acid sequence defined in SEQ ID NO:1.

In the EPO of the present invention also includes a polypeptide having an amino acid sequence identical to at least 70% amino acid sequence constituting EPO, is presented in SEQ ID NO:1.

Identity to the polypeptide sequence of the EPO amino acid sequence defined in SEQ ID NO:1 is preferably not less than 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%.

Alternatively, the EPO may be a polypeptide, which has been replaced, demeterova, inserted and/or added one or more amino acids, provided that the hematopoietic activity is not lost. The number of such changes, preferably not more than 50, more preferably does not exceed 40, even more preferably not more than 30, even more preferably not greater than 20 and most preferably not greater than 10.

As for the method of producing EPO, in addition to recombinante produced EPO, which is produced by using a public animal cells (e.g. Cho cells), prokaryotic organisms or yeasts as hosts, can�e used EPO, as those derived from a natural source and those that are produced in recombinant animals, although not limited to these ERS. Can also be used transgenic birds as recombinant animals that produce EPO. Appropriate is either a purified product, or a product in the raw state, although the purified product is preferred from the viewpoint of quality control.

Purification of EPO can be performed using common methods for proteins such as salting out, adsorption chromatography column, ion-exchange chromatography column, gelfiltration chromatography on a column within the columns methods using antibodies, either individually or in combination, although not limited to these methods. Examples of adsorption chromatography on a column include chromatography on blue sepharose and chromatography on heparin-sepharose. Examples of ion-exchange chromatography on a column include cation exchange chromatography and anion exchange chromatography.

EXAMPLES

The present invention is described in detail in the following examples, although the present invention is not limited to these examples. Except as otherwise agreed to, at the mention of trade names followed the instructions in�robodialer the user guide.

Synthesis originating from cats erythropoietin was performed using the process described in JP-A 2007-89578. The stages of this process are set out again below.

Example of preparation 1

Microinjection of retroviral vector in chicken embryo and artificial breeding chickens in the incubator

Retroviral vector for expression originating from cats erythropoietin was micronational in the chicken embryo, thereby obtaining a transgenic chicken that expresses what is happening from cats erythropoietin. Microinjection and artificial breeding chickens in the incubator was carried out in aseptic conditions. Fertilized chicken eggs (available in the market from Shiroyama Shukeijo) disinfecting the outside of the disinfectant (available on the market from Showa Furanki) and ethanol. Incubator (model P-008(B), available on the market from Showa Furanki) is installed on the operating conditions: 38°C and 50-60% humidity, and since the time of the beginning of incubation (0 hours), which is the time at which included an incubator, incubation was performed at egg turning by 90° every 15 minutes from that time.

After the passage of about 55 hours from start of incubation, eggs were removed from the incubator, the pointed ends cut in the shape of a circle with a diameter of 3.5 cm using a rotary cutting Ministerstva (available on the market from Proxxon), with�Abdenago diamond tip (diameter of the tip 20 mm; the diameter of the shaft 2.35 mm). The contents of the fertilized eggs were transferred into the egg shell, cooked by cutting pointed ends dvuhgolosyj chicken eggs (available in the market from Shiroyama Shukeijo) up to a diameter of 4.5 cm and drop content, and embryos were raised with the help of a syringe plunger. The virus solution was poured into Femtotips II (available on the market from Eppendorf) under the control stereomicroscopes system (SZX12, available on the market from Olympus Corporation), and approximately 2 μl of a solution containing retroviral vector for expression originating from cats erythropoietin mentioned in JP-A 2007-89578, micronational using a FemtoJet (available on the market from Eppendorf).

Using egg white as glue, and each of these holes was sealed with a piece of tape for wrapping products (stock market) from Asahi Kasei Corporation), carved with the receipt of approximately 8×8 cm2after which the eggs were returned to the incubator, and incubation was continued. Turning the eggs in the incubator changed by 30° every 30 minutes. At day 20 from the beginning of incubation in the film for wrapping products made using 20G needles for syringe approximately 20 holes, and incubation is then carried out when applying to the incubator 60 cm3/min of oxygen. Once the Chicks in the eggs began to arise, shell smash, allowing C�the chicken to hatch. Appeared the chickens fed and allowed them to grow. As food used SX Safety and Neo-17 Safety for young chickens (available on the market from Toyohashi Feed Millis Co., Ltd.). The presence originating from cats erythropoietin in blood and eggs of transgenic chickens confirmed the later analysis of cell proliferation using BaF/EPOR.

Example preparation of 2

Cleaning descended from cats of erythropoietin from the egg white

Gathered eggs from individual chickens, egg white which was confirmed by activity originating from cats erythropoietin. Using described later column, originating from cats erythropoietin obtained from egg whites and subjected to cleaning.

All you make to the column, the samples were subjected syringe filter immediately prior to use, using a Millex-HV, pore size of 0.45 μm (available on the market from Nihon Millipore). In case associated with difficulty filtering was carried out by filtration using a Puradisc 25 with a pore size of 2 μm (available on the market from Whatman Ltd.), then carried out the filtration by using Millex.

The definition of the content originating from cats erythropoietin in the treatment process was carried out using the system Biacore 3000 (available on the market from GE Healthcare Japan, BIACORE). Monoclonal antibodies derived from h�man erythropoietin (available on the market from R& D Systems) were subjected to immobilization using N-hydroxysuccinimide on a sensor chip CM 5 quality suitable for research (available on the market from GE Healthcare Japan, BIACORE), using a set of combinations with amines (available on the market from GE Healthcare Japan, BIACORE) for the formation of chips to determine, and then determined the concentration using Epogin as a standard substance and using the program to analyze the system.

To make egg protein on the column was performed pre-treatment to reduce the viscosity. Eggs that were stored in a cold place, returned in the conditions of room temperature and opened, and were separated using egg shells, etc., egg yolk and egg white, then only the egg white was collected and weighed. Egg white stirred using a mixer, thereby reducing the viscosity of the egg white, then added five times the amount of ultrapure water and continued stirring. the pH of a solution of egg albumen during this time approximately from 9.0 to 9.3. To bring the pH to 5.0 was added the appropriate amount of 1N HCl, and stirring is carried out for at least 15 minutes, after which separation was performed by centrifugation at 9500g and 4°C. Then the supernatant was added 1 M NaOH, realizing Samantabhadra pH to 7.0, and then was added 1 M Tris-buffer (pH 7.0) to a final concentration equal to 50 mm. The maximum yield occurring from cats erythropoietin at this stage was 95%.

Then was carried out by chromatography on blue sepharose. In a 50 ml column of Blue Sepharose 6 Fast Flow (available on the market from GE Healthcare Japan, Amersham), balanced with the use of 50 mm Tris (pH 7.0) was added with 500 ml of pre-treated solution of egg white (equivalent to 2-3 egg whites). The column was thoroughly washed with 50 mm Tris (pH 7.0), and then elution was performed using 200 ml of 1 M NaCl and 50 mm Tris (pH 7.0). The resulting elution fraction was subjected to dialysis overnight in a standard way using 20 mm MES (pH 6.2) in a room with low temperature at 4°C, thereby effecting a buffer exchange. The maximum yield occurring from cats erythropoietin at this stage was 98%.

Then was carried out by chromatography on heparin-sepharose. The resulting elution with blue sepharose fraction (after dialysis) was added in two parts on column (HiPrep 16/10 Heparin FF (available on the market from GE Healthcare Japan, Amersham), balanced with the use of 20 mm MES (pH 6.2), and the column was thoroughly washed with each 20 mm MES (pH 6.2), and then was carried out by gradient elution until equal to 80 mm NaCl concentration. The regeneration column is p�ulali each time using 1 M NaCl and 0.1 M NaOH. Collected fractions which are derived from the presence of cats erythropoietin was confirmed using the Biacore system. The maximum yield occurring from cats erythropoietin at this stage was 80%.

Then was carried out by buffer exchange using the column for desalting. The resulting elution from heparin-sepharose fractions were concentrated to a total volume of approximately 30-40 ml using a Vivaspin 20 (available on the market from Sartorius Mechatronics Japan) with a cut off molecular weight of 5,000, to the column for desalting HiPrep 26/10 (available on the market from GE Healthcare Japan, Amersham), equilibrated using 25 mm Tris (pH 7.0) were applied to 10 ml at a time, and elution was carried out using the same buffer, and obtained a fraction containing the protein. the pH was adjusted to 9.0 using 1 M NaOH, in addition to which electrical conductivity was adjusted to 3.0 and 3.2 MS/cm with 1 M NaCl. The maximum yield occurring from cats erythropoietin at this stage was 95%.

Then was carried out by anion exchange chromatography on the column. The sample after the buffer exchange was made in two parts in 5-ml column of HiTrap DEAE FF (available on the market from GE Healthcare Japan, Amersham), balanced with the use of 25 mm Tris (pH 9,0) and electrical conductivity of 3.0 and 3.2 MS/cm, and collected passed through the column without adsorption fraction of each part. To�the PMCs regenerates each time using 1 M NaCl. Fractions were concentrated to a total volume of about 2-3 ml using Vivaspin 20 c cut off molecular weight of 5000. The maximum yield occurring from cats erythropoietin at this stage was 92%.

Then was carried out by gel-filtration chromatography. Concentrated sample was placed in column (Superdex 200 10/300 GL (stock market) from GE Healthcare Japan, Amersham), balanced using 50 mm borate buffer (pH 9,0) or other suitable buffer, and using the same buffer, elution was carried out. Collected fractions which are derived from the presence of cats erythropoietin was confirmed using the Biacore system, and then concentrated to a total volume of about 1-2 ml using a Vivaspin 6 c cut off molecular weight of 5000. The maximum yield occurring from cats erythropoietin at this stage was 93%.

Electrophoresis in SDS-page was performed on the fractions obtained at stages of treatment. The samples were subjected to electrophoresis in denaturing conditions, using a 12.5% e-PAGEL, and were detected using the dye Kumasi Bio-Safe (available on the market from Bio-Rad Laboratories).

Analysis of cell proliferation of Baf/EPOR was performed as described later by way of what is happening from the cats erythropoietin purified from the egg white. As a result of specific actinomycetales 160000-290000 IU/mg.

Attaching PEG to extend from the cats erythropoietin is as follows.

Example 1

Synthesis reaction originating from cats erythropoietin, which is attached to the PEG (Paglinawan EPO cats)

Using a solution of purified EPO cats (20 mm phosphate buffer, pH=7.0), was added the following reagents PEG:

PEG with unbranched chain having a molecular weight of 5000 (available on the market from NOF Corporation; ME-050HS)

PEG with unbranched chain having a molecular weight of 20,000 (available on the market from NOF Corporation; ME-200HS)

PEG with unbranched chain having a molecular weight of 40,000 (available on the market from NOF Corporation; ME-400HS)

The branched PEG chain having a molecular weight of 20,000 (available on the market from NOF Corporation; GL2-200GS2; the number of branch points: 1).

EPO cats and PEG reagent were mixed together in a molar ratio of 1:5 and then the reaction was carried out in close stirring at 4°C. Because when you add the first PEG is formed mono-Mahilyowskaya form, then di-Mahilyowskaya form, then oligo-Mahilyowskaya shape, the formation of appropriate Paglierani forms was monitored using HPLC (stock market) from Shimadzu Corporation) and the reaction stopped at the stage when they have acquired sufficient quantity. As the column used YMC Pack Diol-200 (normal phase system in the presence of �ince from YMC Co., Ltd.).

Once formed, a sufficient number Paglierani forms, 1/10 quantity of 100 mm solution of glycine was added as an inhibitor to stop the reaction when tight mixing at 4°C for 1 hour, after which the reaction mixture is dialyzed against 50 mm including acetic acid buffer, pH 4.5).

Example 2

Separation and purification Paglierani forms of EPO cats in the purification of the reaction mixture with PEG

For separating and obtaining mono-Paglierani, di-Paglierani and oligo-Paglierani forms, formed by reactions with PEG, and the separation of unreacted PEG and unreacted EPO, carried out the separation and purification using a cation exchange column.

The reaction mixture was subjected to separation and purification (binding: comprising 50 mm acetic acid buffer (pH 4.5); elution: gradient to 1 M NaCl) using a column for cation-exchange chromatography (MacroCap SP, available on the market from GE Healthcare Japan). Collected separately peaks corresponding forms, subjected to elution and release with the use of salt concentrations in the following order: oligo-Paglierani, di-Paglierani and mono-Paglierani shape, and each of them was subjected to dialysis against 20 mm phosphate buffer (pH=7,5), 150 mm NaCl). Thereto was added 0.05% of (about./about.) Polysorbate 80(available on the market from Wako Pure Chemical Industries, Ltd.) as the dispersant, thereby obtaining a sample for injection.

Assessment 1: identifying the activities the EPO cats and Paglinawan EPO cats

Have been identified in vitro activity of various Paglierani EPO cats, synthesized in example 2.

The definition of activities the EPO cats was performed using analysis of cell proliferation (JP-A H10-94393), using cells BaF/EPOR (The Chemo-Sero-Therapeutical Research Institute (Kaketsuken), which are a line of EPO-dependent cells. In the analysis of cell proliferation calibration curve of cell proliferation was established using Epogin (available on the market from Chugai Pharmaceutical Co., Ltd.) as the standard of EPO, and EPO activity of unknown samples was determined on the basis of the calibration curve. The medium for the cells BaF/EPOR used liquid medium RPMI 1640 (available on the market from Nissui Pharmaceutical Co., Ltd.), containing 5% fetal bovine serum (FBS) and 50 units/ml penicillin and streptomycin. During normal culturing cells BaF/EPOR added Epogin to a final concentration of 1 u/ml. In the analysis of cell proliferation was used by cells in logarithmic growth phase.

For analysis of cell proliferation using cells BaF/EPOR first from the medium was removed Epogin. Subjected to culturing cells BaF/EPOR was separated by centrifugation within a period of 5 minutes at 1000 Rev/�in. The supernatant was discarded and 10 ml of medium without Epogin was added to the precipitate, which is suspended in it. The same operation was performed three times, thereby removing Epogin from the environment. Cells were counted and diluted with medium without Epogin to a concentration 55555 cells/ml. of the Diluted suspension was inoculated in the amount of 90 μl into each well of 96-well titration the microplate. Thereto was added Epogin, diluted to components 25, 16, 10, 6,4, 4,0, 2,5, 1,6 and 1.0 u/ml concentrations, in the amount of 10 μl to each well, and the cells are evenly suspended in Epogin (final concentration was Epogin 2,5, 1,6, 1,0, 0,64, 0,4, 0,25, 0,16 and 0.1 u/ml, respectively).

Used in the analysis, the samples were subjected to serial dilution medium in approximately 2-4 times on each stage to get into the range of measurements used for the calibration curve, and then a 10-μl portion of each dilution sample was added to the seeded cells, then the cells are evenly suspended. For each standard or unknown sample measurements performed in three replicates. The cultivation was carried out for two days and to each well was then added 10 μl of a solution of Cell Counting Kit-8 (available on the market from Dojindo Laboratories). Color reaction was performed for 1-4 hours, after which the reaction was terminated by adding 10 μl of 0.1 mol/l hydrochloric acid and determined�and the optical density at 450 nm, using the reader for microplates. An approximate formula was derived on the basis of measurement results for standard samples using a logarithmic approximation. The activity of each sample was calculated based on the obtained approximate formulas.

The results of the measurements are presented in table 1.

Assessment 2: a Study using subcutaneous administration in rats

Prepared in example 2, samples were injected subcutaneously to the rats.

The sample solution was subcutaneously injected in a single dose, of 303 mg/kg, male rats (available on the market from Charles River Japan, SPF, age 7 weeks). Table 2 presents experimental group and the samples used for the introduction.

Dee
Table 2
The sample used for the introductionDose (ág/kg)n (number)Method of administration
The molecular weight and structure of attached molecules (molecules) PEGThe number of attached PEG molecules
Group 1 20 kDa, unbranched chainMono3033Subcutaneous
Group 2Dee3033Subcutaneous
Group 3Oligo3033Subcutaneous
Group 45 kDa, unbranched chainMono3033Subcutaneous
Group 5Dee3033Subcutaneous
Group 6Oligo3033Subcutaneous
Group 740 kDa, unbranched chainMono3033Subcutaneous
Group 83033Subcutaneous
Group 920 kDa, branched chainMono3033Subcutaneous
Group 10Dee3033Subcutaneous
All used PEG available in the market from NOF Corporation.

Assessment 3: Determination of reticulocyte count in rats

Believing that the time immediately before subcutaneous injection is day 0, blood samples were collected from the cervical vein sequentially on days 4, 7, 10 and 14 after subcutaneous injection. Using samples of whole blood, the number of reticulocytes in the peripheral blood was determined (by the method of staining with methylene blue: Laboratory-Network-Systems Inc.). Changes in the number of reticulocytes is shown in Fig.1-4.

Rating 4: the Method of measuring the concentration of erythropoietin, which is attached to the water-soluble long-chain molecule(s)

A solution of erythropoietin, which is attached to the water soluble long�pocetna molecule(s), subjected to dialysis against 20 mm phosphate buffer (pH=7,5). Using a spectrophotometer (Gene Quant pro; available on the market from GE Healthcare Japan, Amersham; code 80-2114-98) and UV cell and using 20 mm phosphate buffer (pH=7,5) as a control, the solution is diluted by phosphate buffer so that the optical density at a wavelength of 280 nm was equal to 0.1.

To the solution was added an equal amount of buffer for sample Laemmli (available on the market from Bio-Rad Laboratories, code 161-0737; to which is added DTT to a final concentration of 350 mm), and carried out thermal denaturation at 95°C for 5 min, after which the resulting solution was quickly cooled on ice to get sample, 2 μl of which is subjected to electrophoresis. Electrophoresis was performed for 80 minutes, using gel e-PAGEL E-R12.5L, available on the market from Atto Corporation), using the system for electrophoresis pageRUN (model AE-6531, available on the market from Atto Corporation), and after establishing the current value of 20 mA in the viscous gel. After electrophoresis, the gel is removed from the Board for gel and immersed for 30 minutes in ultrapure water (Milli-Q, available on the market from Nihon Millipore; MILLIPORE ZMQS7V0T1). Ultrapure water was removed and the gel is fixed for 30 minutes in fixing solution. The fixative solution was removed and a solution for staining; then the gel was immersed in it for not less than 3 hours but less than 6 hours.The solution was removed for staining and then the gel was immersed in fixing solution and washed for 60 minutes with cautious shaking, after which the fixative solution used for washing is removed. Then add a new locking solution, perform a 60-minute washing with gentle shaking.

After two washes, the gel image is fixed by irradiation with ultraviolet rays using system ChemiDoc XRS (Bio-Rad Laboratories). Using the software Quantity One version 4.6 (Bio-Rad Laboratories), measure the fluorescence intensity of bands in the image. After defining the lines using the command Frame Lanes, the content is calculated from the percentage of the intensity of the area displayed on screen using the command Lane Background. In cases where, as stated, there are a lot of bands, the ratio of the fluorescence intensity of the bands with a target molecular weight of the sum of fluorescence intensities of all bands is determined as a percentage.

Below is a used 20 mm phosphate buffer (pH=7,5), the fixing solution and the solution for staining.

(i) 20 mm phosphate buffer (pH=7,5), obtained by dissolving the following reagents in 1 l of ultrapure water:

0.59 g dihydrate sodium dihydrogen phosphate (available on the market from Wako Pure Chemical Industries, Ltd.; 192-0825),

5.8 g of dodecahydrate sodium phosphate dibasic (available on the market from Wako Pure Chemical Industries, Ltd.; 196-02835) and

8.76 g of sodium chloride (available on the market from Nacalai Tesque, Inc.; 31320-05)

The aqueous solution prepared with ultrapure water at the following concentrations:

10% methanol (stock market) from Nacalai Tesque, Inc.; 21915-93), and

7% acetic acid (available in the market from Nacalai Tesque, Inc.; 00212-85)

(iii) Solution for staining

Coloring the proteins in the gel substance SYPRO Ruby (Lonza; No. 50564).

1. Pharmaceutical composition having a hematopoietic effect, including erythropoietin cats, is attached to two or more molecules of polyethylene glycol with an unbranched chain, the amount of the specified erythropoietin is not less than 50% of the total content of erythropoietin and erythropoietin which is the polypeptide of (a) to(C):
(a) a polypeptide having the amino acid sequence defined in SEQ ID NO:1;
(b) a polypeptide which has an amino acid sequence identical to at least 90% amino acid sequence defined in SEQ ID NO:1 and has activity of erythropoietin in the analysis of cell proliferation using cells BaF/EPOR; or
(c) a polypeptide which has one or more substitutions, deletions, insertions and/or additions of amino acids relative to the amino acid sequence defined in SEQ ID NO:1 and has activity of erythropoietin in the analysis of cell proliferation using cells BaF/EPOR.

2. Headlights�aseptically composition according to claim 1, where a subject suitable for the introduction of the composition, is an animal of the family Felidae and/or the family Canidae.

3. Pharmaceutical composition according to claim 1 or 2, which produces hematopoietic effect when administered to animals of the family Felidae and/or the family Canidae, which lasts for not less than seven days.

4. Pharmaceutical composition according to claim 1, which is a solution or gel with a pH of at least 4 but not more than 8.

5. Pharmaceutical composition according to claim 1, wherein not less than one, the point of attachment of the polyethylene glycol is a residue of lysine-78.

6. The pharmaceutical composition of claim 1, which even after repeated administration to animals of the family Felidae does not cause the production of antibodies against erythropoietin.

7. A medicament for the treatment of anemia comprising a pharmaceutical composition according to any one of claims.1-6.

8. Hematopoietic tool including a pharmaceutical composition according to any one of claims.1-6.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology and enterology, and concerns treating anaemia in the patients with celiacia. That is ensured by an oral administration of iron bis-glycinate chelate in the form of an effervescent tablet, semi-solid or liquid dosage form in the range of doses of 5 to 200 mg a day that corresponds to 1 to 40 mg of an iron ion.

EFFECT: invention provides treating anaemia in the patients suffering celiacia by the effective absorption of orally administered iron bis-glycinate chelate.

11 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science and aims at treating and preventing alimentary anaemia in young pigs. A preparation contains an iron dextran complex, nanosized and zero-valent selenium (Se0), vitamin E, vitamin B12 and water in the following proportions, wt %: iron dextran complex 0.0001-80.0, selenium (Se0) 0.0001-5.0, vitamin E 0.0001-20.0, vitamin B12 0.0001-10.0, water for injections - the rest.

EFFECT: using the declared method provides compensating iron deficiency in young pigs, improving animal's growth and development, increasing total immunity and rapid adaptation to the varying ambient environment.

4 tbl, 4 ex, 4 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a triazolopyridine compound of general formula [I] or a pharmaceutically acceptable salt, where the partial structural formula: is a group represented by any of the following formulae: or R1 is (1) a hydrogen atom, (2) C1-6alkyl group, (3) phenyl group or (4) C3-8cycloalkyl group; R2 is (1) a hydrogen atom, (2) C1-10alkyl group, (3) phenyl group, optionally substituted with identical or different 1-3 substitutes selected from the following groupB, (4) C3-8cycloalkyl group, (5) C3-8cycloalkenyl group, (6) thienyl group, optionally substituted with 1 substitute selected from halogen or C1-6alkyl group, (7) phenyl-C1-6alkyl group (wherein phenyl is optionally substituted with different or identical 1-2 substitutes selected from halogen, C3-8cycloalkyl or halogen-C1-6alkyl group) or (8) C3-8cycloalkyl-C1-6alkyl group; R3 is (1) a hydrogen atom, (2) a halogen atom, (3) C1-6alkyl group, (4) phenyl group (6) phenyl-C1-6alkyl group; and each of R4 and R5 are both hydrogen atoms or a group B: (a) a halogen atom, (b) C1-6alkyl group, (c) C3-8cycloalkyl group, (d) cyano group and (e) halogen-C1-6alkyl group. The invention also relates to the specific compounds, a pharmaceutical composition based on the compound of formula [I] and to use of the compound with the formula [I].

EFFECT: obtaining novel triazolopyrine compounds, having inhibitory activity on prolyl hydroxylase and capable of inducing erythropoietin production.

30 cl, 34 tbl

FIELD: veterinary medicine.

SUBSTANCE: method of normalisation of the thrombin time duration in newborn calves with iron deficiency anemia consists in the fact that ferroglukin 150 mg (2 ml) intramuscularly is prescribed to newborn calves with iron deficiency anemia, twice with the interval of 4 days, cresacyne 5 mg/kg per day, including it in the scheme of watering for 4 days, starting simultaneously with the first injection of ferroglukin and gamavit intramuscularly once a day in the morning of 0.05 ml/kg for 4 days, starting simultaneously with ferroglukin and cresacyne.

EFFECT: acceleration of normalisation of thrombin time duration, enables to reduce the risk of vascular complications in newborn calves with iron deficiency anemia, to revitalise the herd, to reduce mortality, to maintain the volume and quality of the meat and dairy products obtained from animals.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science, namely to clinical pharmacology and veterinary therapy. The method consists in administering the complex iron-dextran preparation Ferranimal-75M intramuscularly on the 5th day of calf's life in a dose of 3 ml in a combination with an intramuscular injection of the preparation Hydropeptone in a dose of 10 ml. Ferranimal-75M is injected 10 days later in a dose of 2 ml in a combination with an injection of Hyropeptone 5 ml intramuscularly in different points.

EFFECT: method provides higher antioxidative activity of calf's blood serum, reduced pro-oxidant action of iron and incorporated radionuclides, as well as higher iron accessibility in treating and preventing iron-deficiency anaemia in calves exposed to the chronic incorporated radiation.

FIELD: veterinary medicine.

SUBSTANCE: ferroglyukin is administered to new-born calves with iron deficiency anaemia at a dose of 150 mg (2 ml) intramuscularly, twice with an interval of 4 days. Crezacyne 5 mg/kg per day is included in the watering scheme for 4 days, starting simultaneously with the first injection of ferroglyukin. Gamavit is administered intramuscularly once a day in the morning at a dose of 0.05 ml/kg for 4 days, starting simultaneously with ferroglyukin and crezacyne.

EFFECT: method enables to normalise consistently the platelet activity in new-born calves with iron deficiency anaemia during a short period of exposure, transferring it to the level typical of healthy calves, after 4 days of treatment, and to provide long-term maintenance of platelet haemostasis in the optimal mode of operation, eliminating the risk of thrombotic complications in animals and contributing to their normal growth and development.

1 ex

FIELD: metallurgy.

SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.

EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).

17 cl, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to benzoimidazole derivatives of formula (I), as well as to their enantiomers, diastereoisomers, racemates and pharmaceutically acceptable salts, where n equals from 2 to 4, each of R1 substituents is independently selected from H, halogen, -C1-4alkyl, -C1-4pergaloalkyl, trifluoro-C1-4alkoxy, -NO2, -CN, CO2H, -OC1-4alkyl, -SC1-4alkyl, -S(C1-4alkyl)-Rc, -S(O)2(C1-4alkyl)-Rc, -S(O)-C1-4alkyl, -SO2-C1-4alkyl, -S-Rc, -S(O)-Rc, -SO2-Rc, -SO2-NH-Rc, -O-Rc, -CH2-O-Rc, -C(O)NH-Rc, -NRaRb, benzyloxy, phenyl, optionally substituted with one-two Rd, cyanobiphenyl-4-ylmethylsulpfanyl, cyanobiphenyl-4-ylmethanesulphonyl, or -S-(CH2)2-morpholine and two adjacent groups R1 can bind with formation of an aromatic 5-6-membered ring, optionally substituted with one methyl group or two atoms of halogen, optionally containing one or two S or N; Ra and Rb each independently represents C1-4alkyl, -C(O)C1-4alkyl, -C(O)-Rc, -C(O)CH2-Re, C1-4alkyl-Re, -SO2-Rc, -SO2-C1-4alkyl, phenyl, benzyl; or Ra and Rb together with a nitrogen atom, which they are bound with, form a monocyclic 5-6- membered heterocycloalkyl ring, optionally containing one heteroatom, selected from O; Rc represents -C3-8cycloalkyl, phenyl, optionally substituted with one-two Rd, benzyl, optionally substituted with one-three Rd; morpholine; Rd independently represents halogen, -OH, -C1-4alkyl or -C1-4perhalogenalkyl, trifluorine C1-4alcoxy, -OC1-4alkyl, or -O-benzyl optionally substituted with halogen, Re represents -C6heterocycloalkyl, optionally containing one or two of O or N atoms, optionally substituted with a methyl group; R2 and R3 both represent H, -CF3 or C1-3alkyl; each of Z represents a C or N atom, on condition that simultaneously not more than two Z represent N. The invention also relates to particular compounds, a pharmaceutical composition, based on formula (I) compound or a particular said compound, a method of treating diseases, mediated by propyl hydroxylase activity.

EFFECT: novel derivatives of benzimidazole, possessing an inhibiting activity with respect to PHD are obtained.

11 cl, 1 tbl, 186 ex

FIELD: biotechnologies.

SUBSTANCE: in a compound of formula ,

X means N or CH, R1 means hydrogen or cyano, R2 means saturated 4-7-membered residue of heterocyclyl, which is bound through a nitrogen atom that contains 1 to 2 heteroatoms chosen from N and O. Besides, heterocyclyl residue can be replaced with one substituent chosen from a group consisting of C3-C6-cycloalkyl, or with 1-4 fluorine atoms. The invention also refers to a method for obtaining compounds and to a medicine on their basis.

EFFECT: compounds can be used for production of a medicine suitable for being used in a method of treatment or prophylaxis of cardiovascular diseases, cardiac insufficiency, anemia, chronic diseases of kidneys and kidney failure.

16 cl, 1 tbl, 29 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and aims at the simultaneous control of coccidiosis and iron deficiency. The compound comprises triazinones of formulas (I) or (II) wherein R1 means CF3-SO2- or CF3-S-, R2 means CH3, each R4, R5 and R6 means Cl (chlorine) or pharmaceutically acceptable salts thereof, and iron compounds (3+) specified in a group consisting of: multinucleated complex iron (III) and polysaccharide compounds and ammonium-iron (III) citrate. What is also declared is using the above compounds for producing drug preparations.

EFFECT: using the declared group of inventions is effective for the simultaneous control of coccidiosis and iron deficiency; it is accompanied by no adverse phenomena; the compound ingredients have no negative effects on each other; and their biological activity preserves.

17 cl, 15 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention represents a composition for preventing and treating allergic conjunctivitis and keratoconjunctivitis, containing cromoglicic acid, boric acid and water-soluble polymers specified in a group of: carbomer, hypromellose, macrogol and polyvinylpyrrolidone with the components of the composition taken in specific relations, g in 1 ml of the mixture.

EFFECT: invention provides the better reduction of the inflammatory process and symptoms of the disease; there are also ensured ease of use with a smaller frequency of administration, prolonged action and no side effects.

5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with a crystalloid cardioplegic solution, which contains salt solution, including sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydrogen carbonate, water for injections and a structural analogue of natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=CH3, Y=H, Z=OH. The group of inventions also deals with the crystalloid cardioplegic solution, containing salt solution, including sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydrogen carbonate, water for injections and structural analogue of natural apelin X-Arg(NGY)-Pro-Arg-Leu-Ser-His-Lys-Cly-Pro-Nle-Pro-Phe-Z, where X=H, Y=NO2, Z=NH2.

EFFECT: group of inventions provides the recovery of the coronary flow, cardiac contractile and pump function in case of the reperfusion and the reduction of injury to membranes of cardiomyocytes.

2 cl, 2 dwg, 8 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed is a pharmaceutical, ear, sterile, preservative-free composition in the form of a transparent aqueous solution, containing 0.01-0.025 % of fluocinilone acetonide optionally in a combination with 0.1-0.8% of ciprofloxacin or its pharmaceutically acceptable salt, a non-ionic surface-active substance, a tonicity-regulating agent and a viscosity-increasing agent.

EFFECT: composition is useful for the prevention and/or treatment of ear inflammation, optionally accompanied with a bacterial infection, and for the introduction of a single dose from a package.

15 cl, 8 ex

FIELD: medicine.

SUBSTANCE: invention represents a balanced infusion solution containing sodium, potassium and magnesium chlorides, a solvent and sodium L-arginine succinate of formula: Na+[NH=C(NH2)NH(CH2)3CH(NH2)COOH]+[OOC(CH2)2COO]2-. The ingredients in the solution are found in certain proportions, wt %.

EFFECT: invention provides enhanced detoxification activity, low toxicity and wide range of clinical applications.

11 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to production of medications for treating dermatosis. Medication according to invention, made in form of cream, contains mometasone furoate, preservative, hydrophilic no-aqueous solvent, emulsifying agent of 1st kind, emulsifying agent of 2nd kind, emollient, disodium edetate (trilon B), pH-regulating agent, and purified water in quantities, given in invention formula.

EFFECT: invention can be applied for treating inflammatory diseases and itching in case of dermatosis, yielding to glycocorticosteroid therapy.

9 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to cardiac surgery, and represents cardioplegic solution, which contains sodium chloride - 3.41-3.62, potassium chloride - 1.092-1.156 g, magnesium chloride - 3.190-3.485 g, calcium gluconate - 0.0105-0.0130 g, mannite - 4.365-4.520 g, L-carnosine - 20.1504-24.1650 g, N-acetylcarnosine - 8.056-11.032 g, L-histidine - 0.705-0.820 g, water for injections to 1000 ml.

EFFECT: invention ensures prevention of reduction of amplitude, speed of front and speed of pulse-wave reduction, as well as increase of diastolic pressure in left heart ventricle during reperfusion with preservation of buffer capacity and osmolarity of cardioplegic solution with physiological pH parameters.

4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: composition has an action of the central nervous system; it is presented in the form of a solution, contains glycine, glycerol, a preserving agent and water. The invention also concerns a therapeutic agent and a biologically active addition based on the above composition.

EFFECT: group of inventions provides high bioavailability as the composition is presented in the liquid and ease of dosing.

12 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely represents pharmaceutical formulations containing 9-cis-retinyl esters in a lipid excipient. The pharmaceutical formulations containing 9-cis-retinyl esters are described to be applicable in a retinoid replacement therapy for treating retinal degenerations in individuals.

EFFECT: using the formulations for the retinoid replacement therapy for treating retinal degenerations in individuals.

73 cl, 14 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. What is described is a method for preparing a high-purity drug preparation for treating degenerative-dystrophic diseases of peripheral synovial joints and spinal column. The method provides introducing L-proline, an amino acid in an amount of 10-70 g/l into aqueous chondroitin sulphate solution containing no more than 11.5% of an active ingredient, and filtering the solution at temperature 20-50°C through ZetaCarbon (Cuno) R54SLP, R51SLP, R53SLP or AKS (Pall) - AKS 1, 2, 6, 7 filters.

EFFECT: method provides preparing the high-grade 99% pure liquid or lyophilised chondroprotective preparation stabilised with L-proline with the residual content of organic purities: protein - no more than 0,2%, lipids (per 1mg of chondroitin sulphate) - no more than 0,05 mcg, bacterial endotoxins (per 1mg of chondroitin sulphate) - no more than 0,05 EU.

6 cl, 3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to a method for Helicobacter pylori eradication of a gastroduodenal zone by a silver nitrate monotherapy consisting in administering an electrolyte solution of silver ions in the concentration of 300 mcg/l in a daily volume of 960 ml, for the first three days - in an amount of 120 ml every 3 h, 160 ml - every 4 hours and for the following three days - every 6 hours in an amount of 240 ml.

EFFECT: achieving the stable eradication of the vegetative and coccal forms of Helicobacter pylori, reducing the length of treatment by the early recovery of the involved gastric and duodenal mucosa.

3 ex

FIELD: medicine.

SUBSTANCE: invention represents a drug preparation for Parkinson disease containing micronised L-DOPA (3-hydroxy-L-tyrosine) as an active ingredient, which represent stable particles containing poly(lactic-co-glycolic acid 50/50 (PLGA 50/50), or poly(lactic-co-glycolic acid 75/25 (PLGA 75/25), or poly(lactic-co-glycolic acid 50/50 with carboxyl group (PLGA-COOH 50/50), or lactic acid polymer (PLA) in an amount of 75.0÷79.0 wt %, D-mannitol in an amount of 7.5÷8.0 wt %, as well as either polyvinyl alcohol (PVA) or Tween 80.

EFFECT: treating Parkinson disease more effectively.

2 cl, 4 dwg, 2 ex

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