RU2121350C1 - Method of preparing an alpha-fetoprotein preparation - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry. SUBSTANCE: alpha-fetoprotein is dissolved and polyglucin or rheopolyglucin at concentration 4-30 mg/ml is added followed by packaging into ampoules or flasks and lyophilic drying. One ampoule has 0.06-0.09 mg alpha-fetoprotein and 4-30 mg polyglucin or rheopolyglucin. EFFECT: simplified process, increased stability of the end product. 1 tbl, 6 ex

Description

 The invention relates to the pharmaceutical industry and the preparation of a lyophilized preparation of alpha-fetoprotein (AFP), belonging to the group of immunomodulators and used as a means for the treatment of cancer.

 AFP - is a fetal glycoprotein mol. weighing about 70,000 Yes. A large number of AFPs are produced by the cells of the liver and abdominal wall of the fetus. The raw material for AFP is abortive blood obtained with honey. abortions from clinically healthy women who underwent a standard examination. Obtained abortive material must be tested for the absence of antibodies to human immunodeficiency virus (HIV) and for the absence of hepatitis B antigen (HBS-Ag).

 Highly purified AFP is obtained from abortive blood by the methods of affinity chromatography and gel filtration. AFP is used predominantly parenterally.

 It is known that AFP aqueous solutions are unstable during storage, therefore, the task of obtaining highly stable dosage forms is relevant.

 There are various ways to increase the stability of drugs and biological products.

 The most effective way to stabilize drugs for long-term storage is to lyophilize them with appropriate additives (Nikitin V.V., Zvyagin I.V. Freezing and drying of biological preparations. M: Kolos, 1971, 344 pp.).

 Since the effectiveness of stabilization by lyophilization depends on the characteristics of the active substance (its resistance to freezing, dehydration, heating, rehydration, oxidation, and other factors), stabilizing compositions are usually multicomponent and may include various carbohydrates, amino acids, proteins, and their hydrolysates , water-soluble polymers, antioxidants and other substances.

 A known method, for example, is the production of ifosfamide leophilisate by freezing the active substance and auxiliary substances, followed by drying (RF Patent N 1836079 class A 61 K 9/14, publ. 23.08.93; BI N 31).

 The known method consists in the fact that an aqueous or aqueous ethanol solution of ifosfamide containing 1 to 13 wt.% Ifosfamide is mixed with 0.1 to 17 wt. including hexite in terms of 1 wt. including ifosfamide, sterilized by filtration, packaged in appropriate containers and freeze-dried at a temperature of -70 to 0 degrees Celsius. As hexite, mannitol, sorbitol, glucite are used. The method improves the quality of the target product by increasing the shelf life and increasing heat resistance.

 The disadvantage of this method is its unsuitability for obtaining a stable preparation of AFP, since the hexites used as a stabilizer do not have sufficient stabilizing ability against AFP.

 The closest to the claimed method, the prototype, is a method for producing an AFP preparation, including extraction of the latter with butyl alcohol from human abortive material, concentrating the extract to an AFP content of 100 μg / ml, its sterilization and packaging. (Pat. RF N 2026688, A 61 K 47/00, 1995).

The drug is stored at -20 ^o C until use. Before use (1 to 2 hours), AFPs are mixed and incubated with doxorubicin-estrone conjugate taken in equimolar ratios. In the known method, AFP is used as a transport protein for prolonging the action of the main component of the complex - doxorubicin. The disadvantage of this method is its duration, complexity and unsuitability for obtaining a stable preparation of AFP.

 An object of the invention is to simplify the method and increase the stability of the target product.

The problem is achieved by the proposed method, which consists in the following:
The active substance AFP is dissolved in distilled water or physiological saline in an amount of 0.06-0.09 mg / ml, then a structure-forming agent and a stabilizer are introduced, mainly reopoliglyukin or polyglucin in an amount of 4-30 mg / ml, the resulting solution is sterilized by filtration, packaged in ampoules or vials and freeze-dried. Lyophilization is carried out on a TG-50 installation (Germany) for 48 hours with a discharge of 200 to 325 microns of mercury at a temperature of -30 to +25 degrees Celsius.

 An AFP preparation is obtained containing 0.06-0.09 mg of AFP and 4-30 mg of the corresponding polysaccharide in 1 ampoule.

 Polyglukin (m. Weight 30 - 70 kDa) is used in medicine to maintain blood viscosity, is available in the form of a 6% solution.

 Reopoliglyukin (m. Weight 30 - 40 kDa) is used in medicine as a plasma substitute, is available in the form of a 10% solution.

 Unexpectedly, it turned out that these polysaccharides are the optimal excipients for the preparation of the AFP dosage form, since they simultaneously serve as both a stabilizer and a builder. Obtained by the proposed method, the preparation of AFP (conventional name Alfetin) is a porous mass, in the form of tablets from white to white with a grayish tint.

 It is soluble in water or 0.9% sodium chloride solution. The contents of the ampoule dissolve in 0.5 - 1 minute and do not lose activity during storage. Shelf life 2 years at a temperature of no higher than 20 degrees Celsius.

 The defining significant difference of the proposed method from the prototype is the introduction of a stabilizer and a structurant into the AFP solution, which is used as a polysaccharide from the group of dextrans, mainly reopoliglyukin or polyglukin in an amount of 4-30 mg / ml, which improves the stability of the target product during storage.

 At the same time, the lower limits of the amount of stabilizer (4 mg / ml) are determined by the minimum solids content at which the material (preparation) retains the tablet form during lyophilization (IA Muravyov. Drug technology. 1980). The upper limit of the amount of stabilizer is limited by the viscosity of the AFP solution obtained after dissolving the lyophilisate. It has been experimentally established that the use of a polysaccharide in an amount greater than 30 mg / ml is impractical due to the significant viscosity of the AFP solution, unsuitable for injection.

The table presents data on the storage of freeze-dried AFP with various stabilizers. The content of AFP in all experiments was 75 μg / ml. As a stabilizer and structurant used:
1) reopoliglyukin in an amount of 4 mg / ml;
2) polyglucin - 6 mg / ml;
3) dextran (m. Weight 100 kDa) - 5 mg / ml;
4) sucrose ("ChP") - 50 mg / ml;
5) glucose (Farm) - 50 mg / ml;
6) phosphate-saline solution - 0.01 M Na-phosphate buffer pH 7.2 0.2 M NaCl (SDF).

 An AFP preparation without a stabilizer with an active substance content of 75 mg / ml was obtained as a reference standard.

 The table shows that the AFP preparations obtained by the proposed method (approx. 1-3) have good re-solubility and stability during storage. Preparations (approx. 4-6), where disaccharides or SDF are used as stabilizer, have worse solubility and stability during storage. And, finally, the AFP preparation obtained by lyophilization without additives as a stabilizer of reopoliglukin or polyglucin has poor solubility and reduced storage stability.

 It is known to use various carbohydrates as cryo- and xeroprotectors in the lyophilization of various drugs (1 - 3). However, the use of such polysaccharides as reopoliglyukin and polyglukin as structure-forming agents and stabilizers simultaneously during lyophilization of a highly purified specific protein - AFP - is a new, previously unknown quality. The predominant use of the above polysaccharides is determined by their availability (produced by the domestic industry), the permissibility of use in medicine and reasonable price. You can use imported dextrans, for example dextran with m. Weight. 32.60 and 100 kDa are manufactured by Serva, but their use is limited by economic factors.

 Due to the fact that in the well-known sources of a similar method for producing an AFP preparation using a polysaccharide from a group of dextrans as a stabilizer and structure-forming agent, it is not possible to conclude that the claimed method meets the criteria of the invention of "novelty".

 Examples of specific performance.

 Example 1

 To prepare the AFP preparation, 750 mg of AFP and 50 g of reopoliglukin are dissolved in 1 liter of water with stirring, then adjusted to 10 liters with water. The resulting solution is subjected to sterilizing filtration through a membrane filter with a pore size of not more than 0.22 microns, Packed in 10,000 ampoules or vials of 1 ml (single dose) and freeze-dried under aseptic conditions. One ampoule (vial) contains 75 μg of AFP and 5 mg of reopoliglyukin. Each batch of the drug is checked for compliance with the quality requirements of the target product.

 The drug is completely dissolved in 1 ml of water or 0.9% NaCl for 0.5 min.

 The protein content in 1 dose (ampoule) is 75 μg (according to the Lowry method).

 The specific activity of the drug is determined by the amount of AFP contained in it, the determination of which is carried out by enzyme-linked immunosorbent assay (ELISA) using commercial sets of reagents or test systems to determine the concentration of AFP.

 The content of AFP in 1 dose (ampoule) is 75 mcg. The authenticity of the drug is determined by the results of immunochemical and electrophoretic identification of AFP and electrophoretic assessment of the purity of the drug.

 The drug is tested for the absence of impurities of human serum proteins by dot blotting using antibodies against human serum proteins.

 Example 2

 The method is carried out analogously to example 1, but 750 mg of AFP and 50 g of polyglucin are dissolved in 1 liter of water. A preparation is obtained containing (in an ampoule) 75 μg AFP and 5 mg polyglucin.

 The drug dissolves within 0.5 minutes, maintains stability for 2 years.

 Example 3

 The method is carried out analogously to example 1, but 600 mg of AFP and 100 mg of reopoliglyukin are dissolved in 1 liter of 0.9% NaCl. After lyophilization, a preparation is obtained containing 60 μg of AFP and 10 mg of reopoliglukin in 1 ampoule. The drug is dissolved within 1 minute. Maintains stability for 2 years.

 Example 4

 The method is carried out analogously to example 1, but 700 mg of AFP and 70 g of polyglucin are dissolved in 1 liter of water, adjusted to 10 liters with water. A preparation is obtained containing in 1 ampoule 70 μg AFP and 7 mg polyglucin. The drug dissolves within 0.5 minutes, maintains stability for 2 years.

 Example 5

 The method is carried out analogously to example 1, but 900 mg of AFP and 300 g of reopoliglyukin are dissolved in 1 liter of 0.9% NaCl. A preparation is obtained containing in 1 ampoule 90 μg AFP and 30 mg reopoliglukin. The drug dissolves within 1 minute. Maintains stability for 2 years.

 Example 6

 The method is carried out analogously to example 1, but 800 mg of AFP and 80 g of polyglucin are dissolved in 1 liter of water, then the volume of the solution is adjusted to 10 liters. A preparation is obtained containing 80 μg AFP and 8 mg polyglucin in 1 ampoule. The drug dissolves within 0.5 minutes and remains stable for 2 years.

Using the proposed method will allow compared with the prototype
increase the stability of the AFP preparation during storage,
to improve the re-solubility of the drug in water or saline for injection,
to provide the possibility of obtaining a stable tablet-like porous mass, to exclude the loss of the drug when opening the ampoules.

Sources of information
1. Nikitin V.V. Zvyagin I.V. Freezing and drying of biological products. M .: Kolos, 1971, 344 p.

 2. Dolinov K. E. Fundamentals of the technology of dry biological products - M .: Medicine, 1969, 231 p.

 3. RF patent N 2022559, MKI 5 A 61 K 35/54 "Method for the preparation of oncovarial alpha-1-globulin preparation", publ. 11/15/94, Bull. N 21.

 4. RF patent N 1836079, MKI 5 A 61 K 9/14 "Method for producing lyophilisate", publ. 08/23/93, Bull. N 31.

 5. PR N 7 for the production of the drug alpha-fetoprotein for injection, approved. 1995 year

 6. Muravyov I.A. Drug Technology, 1980

Claims (3)

 1. A method of producing an alpha-fetoprotein preparation, characterized in that the alpha-fetoprotein is dissolved, the polysaccharide is introduced in an amount of 4-30 mg / ml, and it is packed in ampoules or vials and lyophilized.  2. The method according to claim 1, characterized in that as a polysaccharide use reopoliglukin or polyglucin.  3. The method according to claim 1, characterized in that when filling the ampoules, the solution is placed in an amount that ensures that the lyophilized preparation contains 0.06-0.09 mg alpha-fetoprotein and 4-30 mg reopoliglukin or polyglucin per ampoule.

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