RU2182830C1 - Tablet form of recombinant human erythropoietin - Google Patents

Abstract

FIELD: pharmacology. SUBSTANCE: tablet administerable per os has phosphate-salt buffer, saccharose, gelatose, peptone, lactose, calcium stearate and erythropoietin. Tablet is covered with acid-resistant shell manufactured from acetylphthalylcellulose. EFFECT: stable and harmless form; retained specific activity in long term storage. 3 tbl

Description

 The invention relates to pharmacology and medicine, and in particular to the preparation of a stable stable recombinant erythropoietin preparation that can be used to treat various anemic conditions.

 Erythropoietin, a glycoprotein hormone, as a physiological regulator of erythropoiesis, determines the number of red blood cells in the peripheral blood of mammals. Every year in clinical practice, the spectrum of anemic conditions is expanding, for which a pronounced therapeutic effect of exogenous recombinant erythropoietin has been identified. However, parenteral administration of the drug, along with the possibility of infection and invasiveness, also causes a number of side effects, due in particular to hypertension due to a sharp increase in red blood cell mass and blood viscosity. Thus, the urgent task of creating an oral alternative form of erythropoietin, which would be devoid of these shortcomings and, judging by the experimental data [1], would change the pharmacodynamics of the drug in the direction of a smoother and more prolonged effect.

 Erythropoietin is quite unstable and can lose its activity both at the stages of creating the finished form of the drug and during storage as a result of exposure to environmental factors. In order to minimize the loss of hormone activity, various combinations of stabilizers are used, which are polymer compounds, proteins and amino acids, sugars, thiol reducing agents, mineral and organic salts. However, many erythropoietin preparations, acquiring sufficient resistance (according to the results of in vitro testing), lose their specific activity in in vivo experiments.

In domestic practice, a number of stable aqueous and lyophilized preparations of erythropoietin have been developed. One of them is an injected stabilized aqueous solution of the hormone containing as a stabilizer dextran with a molecular weight of 30,000-60000 at a concentration of 6-10%, as well as sodium citrate and citric acid to create isotonia and a solution pH of 6.5-7.5. This drug remains active for up to 2 years when stored in conditions of 2-8 ^o C [2].

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

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

This form of erythropoietin, after stabilization and obtaining the final form, retains its specific activity in in vivo experiments, however, by the fifth month of storage at a temperature of +4 - 10 ^o С, the activity loss according to the ELISA readings is 36% of the initial level, with accelerated storage at room temperature activity drop reaches 50%.

 The objective of the invention is the creation of a stable and safe oral form of erythropoietin, which retains specific activity in experiments in vivo, as well as during long-term storage.

The problem is solved by using a tablet form of recombinant human erythropoietin for oral use, including an acid-resistant shell, which is used as acetylphthalyl cellulose, and a core containing a purified substance of erythropoietin in a dose of 100-2000 PIECES per tablet, phosphate-buffered saline; stabilizers: sucrose, gelatose, peptone; fillers: lactose, calcium stearate in the ratio,%:
Phosphate-saline buffer 0.025M - 25-50
Gelatosis - 2-7
Sucrose - 2-7
Meat peptone - 3-7
Calcium Stearate - 1
Lactose - The rest is up to 100
Erythropoietin in a dose - 100-2000 units
As a result of the use of a combination of stabilizers: gelatose 2-7%, sucrose 2-7%, meat peptone 3-7%, it is possible to obtain a stable and safe oral form of erythropoietin, which retains specific activity in in vivo experiments, as well as during long-term storage.

 The decisive factor in this complex of stabilizers is the use of meat peptone, which, in combination with its other components, determines the antioxidant properties of di-, tripeptides [5]. In addition, meat peptone, in contrast to native protein preparations used for stabilization, is not an allergen, cannot be contaminated by viruses and does not contain active inhibitors of erythropoiesis.

 To obtain the drug, a recombinant erythropoietin substance is prepared by collecting the accumulation medium during the cultivation of transformed cells of the erythropoietin producer strain, clarification by filtration of the culture fluid, concentration on hollow fibers from 1:50 to 1: 100 and purification; the concentration of the active material is determined by enzyme-linked immunosorbent assay. In accordance with the desired activity of the finished tablets, the concentrate is adjusted to the required volume with phosphate-buffered saline and combined with the above stabilizers. The resulting liquid semi-finished product is poured under aseptic conditions and subjected to freeze-drying. When lyophilization of the drug, the required amount of solids should be in the range of 7-15%. The freezing – drying regime for the gelatose – sucrose stabilizer system (1: 1 ratio, m / m) was selected earlier on the basis of studying the physical state diagram of the gelatose – sucrose – water system [6].

 The tablets are compressed from a mixture of dry semi-finished product with fillers lactose and calcium stearate. To reduce the hygroscopicity of the tablets, which also affects the stability of the hormone, the lactose content in the final form of the drug is adjusted to 50-70% (the weight ratio of the filler to the lyophilized semi-finished product should be in the range from 1: 1 to 2: 1). The finished tablets are coated with an acid-resistant film based on acetylphthalyl cellulose, which protects the active principle of the drug from the effects of gastric juice.

Thus, the use of meat peptone as a stabilizer, the selection of the necessary ratio of stabilizers, fillers and active component in the tablet allowed us to create a new tablet form of erythropoietin, the stability of which remained for 3 years when stored at a temperature of + 4 ^o C. The biological activity of the tablets was determined both in in vitro experiments (on myelocaryocyte cell culture) and in vivo (oral administration to mice). Moreover, the test results did not reveal differences in colony-stimulating activity between the studied erythropoietin preparation and the commercial Recormon (Table 2).

 When administered to mice, the tablet preparation has a highly specific activating effect on the erythroid germ of the bone marrow, only in the early stages inferior to the analogous one when the injection drug is administered (Table 1). When administered subcutaneously to animals at a dose of 2000 IU / kg, the tablet preparation does not have a toxic effect.

 Example 1. Preparation of erythropoietin lyophilisate for a series of 100 tablets with an activity of 250 PIECES / tablet.

 Pre-prepared, concentrated and purified substance of recombinant erythropoietin is subjected to sterilizing filtration (Millipore 0.22), activity is determined by ELISA.

 Gelatose is obtained by thermal hydrolysis of gelatin in a single excess atmosphere for 3.5 hours

Solutions for the preparation of liquid semi-finished erythropoietin:
FSB (phosphate buffered saline) 0.025M sterilized by autoclaving - 24 ml
50% sucrose solution on FSB, sterilized by filtration - 18 ml
25% gelatin solution on FSB sterilized by autoclaving - 38 ml
25% peptone solution, sterilized by autoclaving - 15 ml
Erythropoietin Concentrate (1600 U / ml) - 31 ml
All solutions under aseptic conditions are combined, poured into 10 ml into 50 ml vials and freeze-dried in a lyophilization apparatus under the following conditions:
The main drying:
Freezing time up to -50 ^o С - 18 h
Drying time - 8 hours
Shelf temperature - -20 ^o С
Sample temperature - -35 ^o C
Temperature rise from -20 ^o С to +20 ^o С - Gradually over 18 hours
Additional drying:
Drying time - 5 hours
Sample temperature - +35 ^o
Pressure - 0.00005 atm
Example 2. Preparation of an experimental series of erythropoietin tablets with an activity of 250 PIECES / tablet, weighing 250 mg in an amount of 100 pcs.

The composition of the ingredients for the preparation of tablets:
Erythropoietin Lyophilisate - 12 g
Lactose - 12 g
Calcium Stearate - 0.48 g
All components are mixed. Biconvex tablets are pressed from the resulting mass and coated with a film based on acetylphthalyl cellulose. The residual moisture content of the tablets is 1.7%. The resulting tablets do not contain pathogenic microflora, retain their shape and do not dissolve in water. The drug can be stored in hermetically sealed vials with virtually no loss of activity at +4 ^o C for a long time (more than 3 years). Storage data (in comparison with the prototype) are given in table.3.

Accelerated storage at a temperature of +22 ^o C also did not reveal a significant decrease in the activity of erythropoietin.

 Example 3. The study of the harmlessness of the tablet form of erythropoietin.

 The toxic properties or harmlessness of the drug is studied on white outbred mice aged 2 months and weighing 17-20 g, as well as on outbred guinea pigs aged 3 months and weighing 250-350 g. To study the toxic effect, the contents of 5 tablets of one series triturated under aseptic conditions, dissolved in 5 ml of an isotonic sodium chloride solution, clarified by centrifugation and 5 ml of 2 guinea pigs and 0.2 ml of 5 mice administered subcutaneously.

 The introduction of the drug did not lead to a violation of behavioral reactions, the mass of animals fluctuated within the physiological norm, the body temperature did not change, and an inflammatory reaction was not observed at the injection site.

 The presented results indicate that a single subcutaneous injection of the drug does not have a toxic effect on the animal's body.

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

 The specific activity of the tablet form of EPO in vivo is tested on male CBA / CaLac mice. Animals once a day for 5 days are prescribed one of the following drugs: 1) saline solution orally in a volume of 0.2 ml; 2) placebo (inert tablet excipient) orally in the same volume; 3) tablets of recombinant human erythropoietin (rhEPO) with a substance content of 250 IU of activity of 5 IU / mouse (total dose of 25 IU / mouse; 4) subcutaneous injections of rhEPO (Protein circuit, St. Petersburg) in an equivalent dose. After the course of injections in animals over the next 5 days, determine the number of red blood cells and reticulocytes in the peripheral blood, the total number of karyocytes (OCC) in the bone marrow of the femur, myelogram is calculated.

 The results of the study show that the administration of rhEPO tablets to mice stimulates erythropoiesis in the bone marrow. Throughout the entire observation period (5 days after drug administration), the absolute content of erythrokaryocytes exceeded the initial level by more than 2-5 times, Table 1) the erythropoestimulating activity of the tablet forms of rhEPO in the initial period was inferior to that for the control injection solution due to differences in pharmacokinetics. However, already on the second day after the end of the course of administration, the number of erythroid cells in the bone marrow when using tablets was 500% of the initial level for parenteral administration. In the following periods, the specific activity of rhEPO in the obtained preparation after oral administration did not differ or exceeded the control figures for parenteral administration of the injection drug.

 Subcutaneous injection of the control drug caused a pronounced sharp reticulocytic reaction in the peripheral blood (Table 1), which gradually decreased towards the end of the study. The introduction of rhEPO tablets caused less significant reticulocytosis, however, this indicator increased by more than 50% compared with the initial level. At the same time, the number of red blood cells corresponded to control figures (injection form) and significantly exceeded the initial level, which speaks in favor of accelerated maturation of reticulocytes.

 Thus, this tabletted form of rhEPO at a dose of 5 U / mouse has a highly specific activating effect on the erythroid bone marrow growth.

 Example 5. The study of the specific biological activity of a concentrated stabilized rhEPO solution in vitro.

The erythropoietic activity of the solution (1150 IU / ml) in comparison with the Swedish drug "Recormon" is determined by its ability to stimulate the growth of erythroid colonies of the type CFU-E. The test system is methyl cellulose culture of non-adherent myelocaryocytes (10 ⁵ cells / ml of medium). The test solution is added in various final dilutions. Incubation is carried out in 24-well plates for 3 days in an atmosphere saturated with 3.5% CO ₂ at 100% humidity. Colonies meant aggregates containing at least 50 cells.

 Counting the grown colonies showed a significant excess of their number relative to the control (without EPO) when using both drugs in the entire range of doses studied (Table 2). The dose dependence of the effect was found in the concentration range from 0.5 to 1.5 U / ml.

 Thus, the stabilized rhEPO solution has erythropoietic activity, without revealing any significant difference in the specified parameter from the commercial drug "Recormon".

LIST OF REFERENCES
1. Goldberg E.D., Dygay A.M., Zhdanov V.V. and others. Hemostimulating properties of the tablet form of recombinant human erythropoietin in the experiment. Bulletin of Experimental Biology and Medicine.

 2. RF patent 2128517. A stabilized aqueous solution of erythropoietin. 6 A 61 K 38/22, 9/08. 04/10/99. Bull. 10.

 3. RF patent 2043118. A method of obtaining an injectable form of erythropoietin. 6 A 61 K 38/22. 09/10/95 Bull. 25.

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

 5. Application of the Russian Federation 97108814 from 05.22.97.

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

Claims (1)

A tablet form of recombinant erythropoietin for oral use, including an acid-resistant membrane, which is used as cellulose acetate, and a core containing a purified substance of erythropoietin in a dose of 100-2000U per tablet, phosphate-buffered saline; stabilizers: sucrose, gelatose, peptone; fillers: lactose, calcium stearate in the ratio, wt. %:
Phosphate-saline buffer 0.025M - 25 - 50
Gelatosis - 2 - 7
Sucrose - 2 - 7
Meat peptone - 3 - 7
Calcium Stearate - 1
Lactose - The rest is up to 100
Erythropoietin in a dose - 100 - 2000 units

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