Tableted form of recombinant human erythropoietin


(57) Abstract:

The invention relates to pharmacology and medicine, namely to receive the preformed stable recombinant erythropoietin, which can be used to treat various anemic conditions. Tableted form of recombinant human erythropoietin for oral administration contains phosphate-saline buffer, sucrose, gelatos, peptone, lactose, calcium stearate, erythropoietin. The tablet is covered with acid-resistant shell, which use acetylcellulose. The technical result is the creation of a stable and safe oral form of erythropoietin, preserving the specific activity in experiments, as well as during long-term storage. 3 table.

The invention relates to farmacologia and medicine, namely to receive the preformed stable recombinant erythropoietin, which can be used to treat various anemic conditions.

Erythropoietin, glycoproteins hormone, as a physiological regulator of erythropoiesis, determines the number of erythrocytes in the peripheral blood of mammals. Every Godot the economic effect of exogenous recombinant erythropoietin. However, parenteral administration of the drug along with the possibility of infection and invasiveness also causes a number of side effects, due, in particular, hypertension due to a sharp increase in erythrocyte mass and blood viscosity. Thus, the actual task of creating oral alternative forms of erythropoietin, which would be devoid of these disadvantages and, according to experimental data [1] , would change the pharmacodynamics of the drug in the direction of the smoothed and more prolonged effect.

Erythropoietin is quite unstable and can lose their activity both at the stage of finished product, and during storage due to exposure to environmental factors. To minimize loss of activity of the hormone used different combinations of stabilizers, which are polymeric compounds, proteins and amino acids, sugar, tirinya reducing agents, mineral and organic salts. However, many drugs erythropoietin, acquiring sufficient stability (according to the results of in vitro testing) lose potency in vivo.

In domestic practice has developed a number of stable water and whether the at contains as stabilizer dextran with molecular weight of 30000-60000 at a concentration of 6-10%, as well as sodium citrate and citric acid to create isotone and pH of 6.5-7.5. This drug remains active up to 2 years when stored under conditions of 2-8o[2].

Known injection lyophilisate erythropoietin containing for stabilization of urea in the amount of 10-15 g/l, a mixture of amino acids in the number 4-24 g/l, physiologically suitable buffer and calcium chloride as complexing agents. The specified drug is stable in the atmosphere of nitrogen for 2 years at 0o[3]. However, all the above forms of drugs are not oral.

The closest in composition and method of introduction to the proposed drug is a pill form of erythropoietin [4 prototype], containing as stabilizers to gelatos 3-5%, sucrose 3-5% and urea 1-2%.

This form of erythropoietin after stabilization and end shape retains its potency in vivo, however, by the fifth month of storage at a temperature of 4 to 10oWith the loss of activity by ELISA readings constitute 36% of the initial level, under accelerated storage conditions room temperature drop of activity reaches 50%.

Task izobretatelnosti in vivo, as well as during long-term storage.

The problem is solved by the use of a tablet form of recombinant human erythropoietin for oral administration, including acid-resistant shell, which use acetylcellulose, and the core containing the purified substance erythropoietin at a dose of 100-2000 ED pill, phosphate-saline buffer; stabilizers: sucrose, gelatos, peptone; excipients: lactose, calcium stearate in a ratio, %:

Phosphate-saline buffer 0.025 M - 25-50

Gelatos - 2-7

Sucrose - 2-7

Peptone meat - 3-7

Calcium stearate - 1

Lactose - the Rest is up to 100

Erythropoietin dose - 100-2000 U

As a result of application of a combination of stabilizers: gelatos 2-7%, sucrose 2-7%, peptone meat 3-7% possible to obtain a stable and safe oral form of erythropoietin, preserving the specific activity in vivo, as well as during long-term storage.

Decisive in this complex stabilizers is the use of peptone meat, which in combination with the other components causes the antioxidant properties of di-and tripeptides [5]. In addition, meat peptone, unlike native who moustache and does not contain active inhibitors of erythropoiesis.

To obtain the drug substance recombinant erythropoietin is prepared by collecting the cumulative environment in the process of culturing the transformed cells of strain-producer of erythropoietin, bleaching, filtering the culture fluid, the concentration of the hollow fibers is from 1:50 to 1:100 and cleaning; the concentration of active material define a solid phase enzyme immunoassay method. In accordance with the desired activity of the prepared tablets concentrate is brought to the required volume of phosphate-saline buffer and combined with the above-mentioned stabilizers. The liquid semi-finished product is bottled under aseptic conditions and subjected to freeze-drying. When lyophilization of the drug required amount of dry residue must be in the range of 7-15%. Mode freeze - drying system stabilizers gelatos - sucrose (1:1 ratio, m/m) was selected previously on the basis of studying the chart of the physical States of the system gelatos - sucrose - water [6].

Tablets are pressed from a mixture of dry cake mix with fillers lactose and calcium stearate. To reduce the hygroscopicity of tablets, which also affects the stability of the hormone, lactose is the must is in the range from 1:1 to 2:1). Ready tablet cover acid-resistant film on the basis of acetylcellulose protecting the active principle of the drug from exposure to gastric juice.

Thus, using as a stabilizer peptone beef, selection of the proper ratio of stabilizers, fillers and active component in the tablet helped to create a new tablet formulation of erythropoietin, which stability was maintained during the 3 years when stored at a temperature of +4oC. the Biological activity of the tablets was determined in experiments in vitro (cell culture myelokaryocytes) and in vivo (oral mouse). The results of the test revealed no differences in colony-stimulating activity between the study drug erythropoietin and commercial Recormon" (PL.2).

When administered to mice tablets the drug has a high specific activating effect on erythroid Rostock bone marrow, only in the early stages inferior similar to the injectable drug (table.1). After subcutaneous administration to animals at a dose of 2000 IU/kg tablets the drug is not toxic.

Example 1. Privateline prepared, concentrated and purified substance recombinant erythropoietin is subjected to sterilizing filtration (Millipore 0,22), activity is determined by ELISA.

Gelatos receive as a result of thermal hydrolysis of gelatin at one of the excess atmosphere for 3.5 h

Solutions for cooking liquid semi erythropoietin:

FSB (phosphate-saline buffer) 0.025 M, sterilized by autoclaving - 24 ml

50% sucrose solution on the FSB, sterilized by filtration - 18 ml

25% solution gelatos on the FSB, sterilized by autoclaving and 38 ml

25% peptone solution, sterilized by autoclaving 15 ml

Concentrate erythropoietin (1600 IU/ml - 31 ml

All solutions under aseptic conditions combine, poured into 10 ml vials with a volume of 50 ml and freeze-dried in the freeze-drying under the following conditions:

Primary drying:

Time freezing to -50oC - 18 h

Drying time - 8 hours

The shelf temperature is -20oWITH

The sample temperature is -35oWITH

Temperature rise from -20oC to +20oWith Gradually within 18 hours

Additional drying:

The drying time is 5 hours

The sample temperature is +35o< / BR>
Yu 250 IU/tablet weight 250 mg in quantities of 100 pieces

The composition of ingredients in tablets:

The lyophilisate erythropoietin - 12 grams

Lactose - 12 grams

Calcium stearate - 0,48 g

All the components are mixed. The resulting mass is pressed tablets biconvex shape, and a cover film on the basis of acetylcellulose. Residual moisture tablets of 1.7%. The obtained tablets do not contain pathogenic organisms, retain their shape and do not dissolve in water. The drug can be stored in sealed bottles with virtually no loss of activity at +4oWith long time (more than 3 years). Data storage (in comparison with the prototype) are given in table.3.

Accelerated storage at a temperature of +22oWith also revealed no significant decrease in activity of erythropoietin.

Example 3. Study the safety of a tablet form of erythropoietin.

The toxic properties or harmlessness of the drug is studied in outbred mice aged 2 months and weighing 17-20 g, as well as in outbred Guinea pigs aged 3 months and weighing 250-350 g For studying toxic effects contents 5 tablets one series in aseptic conditions grind, dissolve in 5 the pigs and 0.2 ml of 5 mice.

The drug did not lead to the violation of the behavioral responses, the weight of the animals ranged from physiological norms, body temperature did not change, the inflammatory reaction at the injection site were noted.

The presented results indicate that a single subcutaneous administration of the drug is not toxic to the animal organism.

Example 4. The study of specific biological activity of a tablet form of erythropoietin in vivo.

The specific activity of the preformed shape of EPO in vivo test in mice male CBA/CaLac. Animals 1 time a day for 5 days appoint one of the following drugs: 1) saline orally in a volume of 0.2 ml; 2) placebo (inert filler tablets) orally in the same volume; 3) tablets of recombinant human erythropoietin (rhepo) with substance 250iu activity 5 IU/mouse (total dose of 25 IU/mouse; 4) subcutaneous injection repo ("Protein component", St. Petersburg) in the equivalent dose. After the course of injections in animals over the next 5 days to determine the number of erythrocytes and reticulocytes in the peripheral blood, the total number is of show what purpose tablets repo mice leads to stimulation of erythropoiesis in the bone marrow. During the entire observation period (5 days after drug administration) absolute content eritrotsitov exceeded more than 2-5 times the original level, PL.1) retrobestiality activity tabloids repo in the initial period yielded a similar indicator for the control of injection solution due to differences in pharmacokinetics. However, already on the second day after completion of the course introduction the number of erythroid cells in the bone marrow using tablets was 500% from baseline parenteral purpose. In subsequent periods the specific activity repo received the drug by oral administration was not different or exceeded targets with a parenteral injection of the drug.

Subcutaneous injection control of the drug caused a pronounced sharp reticulocyte reaction in the peripheral blood (table. 1), which gradually decreased to the end of the study. The introduction of tablets repo caused less significant reticulocytosis, however, this figure was increased by more than 50% compared with ishonmasla initial level, what speaks in favor of the accelerated maturation of reticulocytes.

Thus, this preformed shape repo at a dose of 5 IU/mouse provides highly specific activating effect on erythroid Rostock bone marrow.

Example 5. The study of specific biological activity of the concentrated stable solution repo in vitro.

Eritropoeticescoe activity solution (1150 U/ml) in comparison with the Swedish drug "Recormon" determined by the ability to stimulate the growth of erythroid colonies type CFU-E. as the test system is methylcellulose culture of non-stick myelokaryocytes (105cells/ml of medium). The investigated solution add in different final dilutions. Incubation is carried out in a 24-hole tablets for 3 days in an atmosphere saturated 3.5% of CO2at 100% humidity. Under the colonies meant aggregates containing at least 50 cells.

Calculation of grown colonies showed a significant excess of their numbers relative to the control (without EPO) when using both drugs in the whole range of the investigated doses (table. 2). Dose dependence of the effect detected in the concentration range from 0.5 to 1.5 U/ml

Textoverlay difference at the specified parameter from the commercial preparation "Recormon".


1. Goldberg E. D., Digi A. M., Zhdanov Century. Century. and other Gemostimuliruyuschee properties of a tablet form of recombinant human erythropoietin in the experiment. Bulletin of experimental biology and medicine.

2. RF patent 2128517. Stabilized aqueous solution of erythropoietin. 6 A 61 K 38/22, 9/08. 10.04.99. Bull. 10.

3. RF patent 2043118. The method of obtaining injectable form of erythropoietin. 6 A 61 K 38/22. 10.09.95 bull. 25.

4. Steele R. P. M., Davies J. D., Greave J. Hyg., Camb. 1969, 67, 104-114.

5. RF application 97108814 from 22.05.97.

6. Shalaev, E. Y., N. Varaksin A., Rjabicheva T. G., Cryo Letters, 1996, v. l7, 183-194.

Tableted form of recombinant erythropoietin for oral administration, including acid-resistant shell, which use acetylcellulose, and the core containing the purified substance erythropoietin at a dose of 100-ED a tablet, phosphate-saline buffer; stabilizers: sucrose, gelatos, peptone; excipients: lactose, calcium stearate in the ratio, by weight. %:

Phosphate-saline buffer 0.025 M - 25 - 50

Gelatos - 2 - 7

Saccharose - 2 - 7

Peptone meat - 3 - 7

Calcium stearate - 1

Lactose - the Rest is up to 100

Erythropoietin dose - 100 - ed


Same patents:

The invention relates to pharmaceutical compositions that increase fetal hemoglobin of the patient or accelerating differentiation of cells containing an effective amount of butyrate precursor drug of formula I in which a and D are independently from each other selected from the group comprising hydrogen, or C(1-4) straight or branched alkyl, S(2-4), straight or branched alkenyl or quinil, which may be independently substituted by hydroxy, alkoxy, carboxylation, alkylamides and so on, provided that a and D are not both hydrogen, R is oxygen, NH, NC(1-5) alkyl straight or branched chain or NH–°(2-5)of alkenyl straight or branched chain, any of which may be optionally substituted by a residue of carbocycle or heterocycle, Z is hydrogen, C(1-4) alkyl straight or branched chain, With(2-4) alkenyl or quinil straight or branched chain, carbocyclic or heterocyclic residue and so on, and each stereogenic carbon atom can be R or S configuration, and pharmaceutically acceptable adjuvant or carrier
The invention relates to medicine, namely to Pediatrics, and for the treatment of immunopathy in iron deficiency anemia in children residing in a large industrial city with a developed chemical and petrochemical industry

The invention relates to novel acyl derivatives of guanosine formula I, inosine formula II, xanthosine formula III, deoxyinosine formula IV, deoxyguanosine formula V, inosine - 2',3'-(acyclic)dialcohol formula VI or pharmaceutically acceptable salts

The invention relates to medicine, in particular to the production of medical biological preparations, and relates to oral formulations of recombinant human erythropoietin, which is used for the treatment of various forms of anemia

The invention relates to medicine, specifically to antibacterial substance erythromycin

The invention relates to the field of pharmacy

The invention relates to medicine and relates to a pharmaceutical composition having enteral action

The invention relates to pharmaceutical industry and relates to oral dosage forms, soluble in the colon, and a sheath material for her

The invention relates to pharmaceutical industry

The invention relates to the field of pharmacy and for the matrix particles of the medium, solid dosage forms derived from it, and the method of preparation of such matrices media and dosage forms that disintegrate or dissolve within a few seconds, being placed in the oral cavity
The invention relates to clinical Oncology and related medication for the treatment of malignant tumors by the method of photodynamic therapy

The invention relates to genetic engineering and medicine

The invention relates to the meat industry, namely the production of products from the blood of slaughtered animals, as a preventive and curative medicines for children