RU2319479C2 - Method for preparing alpha-fetoprotein preparation tablet - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry.

SUBSTANCE: invention proposes a method for making tablets of alpha-fetoprotein preparation. Method involves mixing alpha-fetoprotein with a stabilizing agent, addition of a filling agent consisting of microcrystalline cellulose and potato starch or lactose, coating the prepared mixture by envelope consisting of acetylphthalylcellulose in the following ratio of components, weight%: alpha-fetoprotein, 0.01-0.03; stabilizing agent, 8.57-14,28; microcrystalline cellulose, 40-45.7; potato starch or lactose, 40-45.7, and acetylphthalylcellulose, 1.14-2.85. Rheopolyglucin is used as a stabilizing agent, and alpha-fetoprotein is obtained from abortive, cord or placental blood by separation of inert proteins from blood by centrifugation, fractionation of supernatant with ammonium sulfate or sodium sulfate, or ethyl alcohol for 1 h, repeated centrifugation of solution, carrying out the first chromatography treatment of supernatant on an affinity sorbent using Sepharose CL-4B, elution of alpha-fetoprotein with 0.1 M glycine-HCl buffer solution at pH 2.5 up to obtaining zero values of optical density at wavelength 280 nm, and the following chromatography treatment on an affinity sorbent using Sepharose CL-4B containing immobilized antibodies against human IgG and dialysis of alpha-fetoprotein solution against 0.15 mole/l of sodium chloride solution. The end product is diluted with rheopolyglucin up to the concentration alpha-fetoprotein 0.9 mg/ml and that of dextran 10 mg/ml. Invention provides increasing yield of alpha-fetoprotein and maximal purity of the end product, increasing stability of alpha-fetoprotein in storage, expanding region of its using and design of preparation of prolonged effect.

EFFECT: improved preparing method.

3 ex

Description

The method is intended to produce an alpha-fetoprotein (AFP) preparation and can be used in the pharmaceutical industry and medicine. The invention relates to a method for producing a tablet form of AFP serum protein, including the isolation of AFP, its stabilization and mixing with excipients, shaping and coating. AFP belongs to the group of immunomodulators and can be used as a means for the treatment of cancer, autoimmune and allergic diseases.

Recent epidemiological data published in a number of reputable scientific publications indicate that high AFP levels in pregnant women reduce their subsequent risk of both pre- and postmenopausal breast cancer and may be associated with the properties of AFPs in relation to suppression of tumor growth. It has been shown that a reduced risk of postmenopausal breast cancer is associated with high levels of AFP in the third trimester in women who had their first pregnancy before 28 years. Subsequent studies have confirmed and expanded the data of previous work by the fact that the values of AFP in the blood of the second trimester in premenopausal women under 38 years of age also contribute to reducing the risk of developing malignant neoplasms. It follows that high levels of AFP in the blood serum of the mother during any pregnancy were associated with a low incidence of breast cancer; the connection was especially strong during pregnancy at a young child-bearing age. These results prompted the assumption that AFP and its secondary peptides can be used not only in cancer therapy, but also to prevent other possible types of tumors. This protective effect of AFP during pregnancy has also been observed in women with multiple pregnancy (twins), neural tube defects, and presentation with preeclampsia. All these situations share a commonality of elevated serum AFP levels, which may contain sufficient amounts of conformationally induced AFP variants to induce suppression of the growth of cancer microfocus present in the mother’s breast tissue (Gerald J. Mizejewski, 2002).

AFP - is a fetal glycoprotein with a molecular weight of about 70,000 D. AFPs are produced in large quantities by liver cells and the abdominal wall of the fetus. Abortive, umbilical and placental blood obtained during medical abortions from clinically healthy women who underwent a standard examination is the raw material for obtaining AFP. The resulting material must be tested for the absence of antibodies to the human immunodeficiency virus (HIV) and for the absence of hepatitis B virus and antibodies to hepatitis C.

AFP is used predominantly parenterally. AFP aqueous solutions are unstable during storage, so the task of obtaining highly stable dosage forms is relevant. The most effective way to stabilize drugs for long-term storage is to lyophilize them with appropriate additives [5].

Oncofetal proteins are obtained from human or animal embryonic or tumor tissue. Highly purified AFP is obtained from abortive blood by affinity chromatography.

A known method of producing AFP from human tumor tissue, including the isolation and chromatographic purification of the target product sequentially on several sorbents [2]. Its disadvantage is the high complexity of the process and the low yield of the target product.

A method has been developed for obtaining AFP from human embryonic tissue [1], including its grinding, washing, and trypsin treatment for 1 h at a temperature of no more than 37 ° C. The resulting cell suspension was cultured in a nutrient medium for 7-8 days at 37 ° C, and the target product was separated by centrifugation at 3000 rpm. A clear orange-yellow liquid is obtained containing 10% AFP.

The disadvantages of this method are the low yield and high content of impurity proteins.

There is a method for producing AFP from human embryonic tissue [2], including processing of the starting material with butyl alcohol, centrifugation, dialysis of the aqueous protein phase relative to a 0.9% sodium chloride solution, centrifugation, incubation of the obtained supernatant with estrogen at a final concentration of 0.005% at a temperature of 0-10 ° C for 8 h and the isolation of the target product from the supernatant by chromatography on sepharose CL-4B with immobilized estrogen. The yield of the target product is 60-70%, and the purity of the drug is 90-95%.

The disadvantages of the method are the lack of purity of the target product, the presence of an admixture of albumin and hemoglobin, as well as the high complexity of the method and the need to work with a flammable sharply smelling organic reagent - butyl alcohol.

One of the closest to the invention is a method for producing AFP from human embryonic tissue of 8-12 weeks, obtained during medical abortion, including the stages [7]: grinding and homogenization of embryonic tissue; centrifuging the homogenate at 5000 rpm; clarification of the supernatant by centrifugation at 12,000 rpm; purification of the target product by chromatography on DE-cellulose; purification of the target product by Sephadex G-100 chromatography with cybacron blue F36-A dye immobilized on it; purification of the target product by rechromatography on Sephadex G-100 with cybacron blue dye F26-A immobilized on it; purification of the target product by chromatography on DE-cellulose.

The disadvantages of the described method are the duration of the procedure, low yield (25-30%) and insufficient purity (85-90%) of the target product, high complexity and the need for sophisticated equipment (high-speed centrifuges).

The closest analogue is a method for producing AFP [4]: human abortive blood is centrifuged, the resulting supernatant is subjected to two-step chromatographic purification (affinity chromatography on Br-CN-sepharose with immobilized antibodies to AFP and affinity chromatography on Br-CN-sepharose with immobilized balanced complex human blood proteins). The resulting AFP solution is desalted by dialysis against water, sterilized and lyophilized.

The disadvantages of the prototype are low yield (60-70%) and high contamination with impurity proteins (purity 90-95%) of the target product.

The defining differences of the proposed method from the prototype are the preliminary purification of the initial blood serum by fractionation of serum proteins with ethyl alcohol or ammonium or sodium sulfates, application to the affinity sorbent in the presence of sodium chloride and X-100 triton to suppress nonspecific sorption and inactivation of viruses and to refine the preparation to a homogeneous state on a second immunosorbent representing sepharose modified with antibodies against human IgG.

A known method of producing AFP tablets is that AFP is isolated from blood serum, mixed with albumin, fillers consisting of microcrystalline cellulose and potato starch or lactose are added, the resulting mixture is formed into tablets and coated with a membrane consisting of cellulose acetate at a certain ratio ingredients (RF patent No. 2154468, IPC А61К 9/20, А61Р 37/00 dated November 9, 1999).

The disadvantages of this method are the use of AFA as a stabilizer of human albumin, which carries additional antigenic load when it enters the human body and instability during storage at various temperature conditions.

An object of the invention is to increase the yield of AFP and achieve maximum purity, increase storage stability, expand the scope, creation of a drug of prolonged action.

The task is achieved as follows.

Checked for the absence of hepatitis B virus, antibodies to HIV, hepatitis C, abortive, umbilical cord or placental blood is purified from impurity proteins (in particular, immunoglobulins) by fractionation with ethyl alcohol (5-30%) or ammonium or sodium sulfate (degree of saturation 30-40 %), the precipitate formed is centrifuged and discarded. The supernatant is lyophilized and / or dialysed relative to a sodium chloride solution (0.15 mol / L) and sterilized by filtration through a cascade of filters with pore sizes of 3.0 to 0.22 microns. Sodium chloride (0.1-1.0 mol / L) and Triton X-100 (0.01-0.5%) are added to diluted sterile serum to block non-specific sorption and inactivation of viruses. Thus prepared sterile serum is applied to a sorbent with immobilized antibodies to AFP. Non-specifically sorbed blood components are washed out from the sorbent sequentially with 4-5 volumes of 0.15 M phosphate-buffered saline pH 7.0-7.5 containing 0.5 M sodium chloride and the same volume of sodium chloride solution (0.15 mol / l). Affinity-bound AFP is eluted with glycine-HCl buffer solution (0.05-0.15 mol / L).

The eluate is neutralized to a pH of 6.0-8.0 and applied to a sorbent with immobilized antibodies against human IgG, where affinity sorption of human immunoglobulins (negative immunosorption) occurs, contaminating the drug after the first affinity purification. AFP obtained after negative immunosorption is diluted with 0.9% sodium chloride solution and reopoliglyukin to a concentration of AFP 0.9 mg / ml and dextran 10 mg / ml. After sterilizing filtration, the AFP substance is mixed with microcrystalline cellulose (MCC), potato starch or lactose, formed into tablets, and then coated with a coating of acetylphthalyl cellulose (AFC), in the following ratio of ingredients, wt.%:

AFP 0.02-0.03 Reopoliglyukin 8.57-14.28 MCC 40.0-45.7 Potato starch or lactose 40.0-45.7 AFC 1.14-2.85

The proposed method is as follows. Prepare a dry mixture in the following ratio:

AFP 0.02-0.03 wt.% 0.10-0.12 mg;

Reopoliglukin 8.57-14.28 wt.% 30-50 mg;

MCC 40.0-45.7 wt.% 140-160 mg;

potato starch or lactose 40.0-45.7 wt.% 140-160 mg.

Mechanical stirring is carried out for at least 1 hour to achieve a uniform composition. The mixture is wetted with acetone (TSP) to a pasty state. Tableting of the resulting mass is carried out at room temperature under pressure. The resulting tablets are dried at a temperature of 37.0-37.5 ° C under a hood. The humidity of the tablets should be no more than 7-8%. The dried tablets are immersed in a solution containing about 5% AFC and mixed for 15 minutes. Then the liquid is filtered off, and the tablets are dried at a temperature of 37.0-37.5 ° C. Coated tablets coated with AFC (4-10 mg) are insoluble at pH 1.0 for 2 hours, lose their shell and dissolve within 30 minutes at pH 8.0, i.e. at pH in the human intestines.

The resulting tablets have a weight of from 314.1 to 380.12 mg.

Examples of specific performance.

Example 1

All procedures are carried out at + 4 ° C. 3 L of blood serum is centrifuged at a speed of 3500 rpm for 1 hour, the ballast sediment is discarded, and saturated ammonium sulfate solution is slowly added to 2.5 L of the supernatant until 33% saturation is reached, incubated for 1 hour and centrifuged at 2500 rpm within 30 minutes 2 l of the supernatant is dialyzed relative to a solution of sodium chloride (0.5 mol / l), diluted with a solution of sodium chloride (0.5 mol / l) to 8 l, add triton X-100 to a concentration of 0.01% and incubated with 1 l of sepharose CL-4B with immobilized antibodies to AFP. Incubation is carried out for 48 hours with constant stirring.

Non-specifically adsorbed blood components are washed from the sorbent sequentially with 4-5 volumes of phosphate-saline buffer solution containing 0.5 M sodium chloride, and the same amount of 0.15 M sodium chloride solution.

After washing the sorbent with a solution of sodium chloride (0.15 mol / L), an elution with 0.1 M glycine-HCl buffer solution with a pH of 2.5 is carried out. The elution process is controlled by optical density at a wavelength of 280 nm. Elution is carried out until the optical density reaches zero. The eluate containing the target product is neutralized to a pH of 6.5-7.5 and applied to a CL-4B Sepharose column with immobilized anti-human IgG antibodies.

The resulting preparation containing AFP, purified from globulins, albumin and other impurity proteins, is dialyzed relative to a solution of sodium chloride (0.15 mol / L), diluted with reopoliglucin to a concentration of AFP of 0.9 mg / ml and dextran 10 mg / ml.

After sterilizing filtration, the AFP solution is poured into vials and stored at + 4 ° C until further process steps. The electrophoretic purity of the obtained preparation is analyzed by electrophoresis in a gradient of polyacrylamide gel. The purity of the obtained AFP is more than 98%. The yield of AFP is 75%. The resulting AFP is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C. The drug is sterile, does not contain pyrogenic and toxic impurities.

Then, to prepare tablets, 0.10 mg of AFP, 30 mg of reopoliglukin, 140 mg of MCC and 140 mg of potato starch are taken, mixed for 1 hour until a uniform composition is achieved. The mixture is wetted with acetone (TSP) to a pasty state. From the resulting mass, tablets are made under pressure at room temperature, they are dried at t 37.0-37.5 ° C under an extract until the tablets reach a moisture content of 7-8%. The dried tablets are immersed in a solution containing about 5% AFC, stirred for 15 minutes, the liquid is filtered off, the tablets are dried at t 37.0-37.5 ° C.

Example 2

All procedures are carried out at + 4 ° C. 3 l of blood serum is centrifuged at a speed of 2500 rpm for 1 h, the ballast sediment is discarded, and saturated ammonium sulfate solution is slowly added to 2.5 l of the supernatant until 33% saturation is reached, incubated for 1 h and centrifuged at 2500 rpm within 30 minutes 2 l of the supernatant is dialyzed relative to a solution of sodium chloride (0.5 mol / l), diluted with a solution of sodium chloride (0.5 mol / l) to 8 l, add newt X-100 to a concentration of 0.01% and applied to a chromatographic column, filled with 1 l of sepharose 4B-CL with immobilized antibodies to AFP. Application rate 320 ml / h. Application is carried out three times the passage of the applied volume through the column.

Non-specifically adsorbed blood components are washed out of the sorbent sequentially with a phosphate-saline buffer solution containing 0.5 M sodium chloride and 0.15 M sodium chloride solution at a rate of 300 ml / h until the optical density reaches zero.

After washing the sorbent with a solution of sodium chloride (0.15 mol / l), an elution with 0.1 M glycine-HCl buffer solution with a pH of 2.5 and a speed of 150 ml / h is carried out. The elution process is controlled by optical density at a wavelength of 280 nm. Elution is carried out until the optical density reaches zero. The eluate containing the target product is neutralized to a pH of 6.5-7.5 and applied to a CL-4B Sepharose chromatography column with immobilized anti-human IgG antibodies. Application rate 80 ml / h.

The entire solution passing through the column containing AFP, purified from globulins, albumin and other impurity proteins, is dialyzed relative to a solution of sodium chloride (0.15 mol / l) and diluted with reopoliglucin to a concentration of AFP of 0.9 mg / ml and dextran 10 mg / ml .

After sterilizing filtration, the AFP solution is poured into vials and stored at + 4 ° C for further process steps. The electrophoretic purity of the obtained AFP preparation is checked by polyacrylamide gel gradient electrophoresis. The purity of the obtained AFP is more than 98%. The yield of AFP is 75%. The resulting preparation is tested for the absence of antibodies to HIV, hepatitis B virus and antibodies to hepatitis C. The drug is sterile, does not contain pyrogenic and toxic impurities.

Further, the cooking method is carried out analogously to example 1, but instead of potato starch take lactose in the same amount.

The composition of the tablets is as follows, mg:

AFP 0.12 Reopoliglyukin 50,0 MCC 160,0 Lactose 160,0 AFC 10.0

Example 3

The method is carried out analogously to examples 1 and 2, but the composition of the tablets is as follows, mg:

AFP 0.1 Reopoliglyukin 40,0 MCC 150.0 Lactose 150.0 AFC 7.0

The use of the proposed method aims to significantly improve the purity of the target product due to preliminary fractionation of the proteins of the initial blood serum and double affinity chromatography on sorbents of high specificity; increase the yield of AFP from an affinity sorbent with immobilized antibodies to AFP due to the use of glycine-HCl buffer solution; reduce the time of the full technological cycle due to the absence of a gel filtration stage; get a convenient, prolonged-acting dosage form of AFP; use AFP tablets not only for therapeutic purposes (immunomodulator and antitumor effect), but also for prophylactic purposes (prevention of tumor formation).

Information sources

1. A.S. No. 403407, class ³ A61K 35/48. "A method of obtaining a biostimulant." Claim 11/26/68. Publ. 10.26.73.

2. A.S. 1497806 USSR, MKI ⁴ A61K 37/02. 48. "Method for producing alpha-fetoprotein."

3. A.S. 2100031. "A method of producing alpha-fetoprotein." / V.V. Starikov, S.Yu. Rodionov, Myakkohodov V.A., Pak N.A. (Russia). - No. 95120418/113. Claim 12/01/95. Publ. 12/27/97. Bull. Number 36.

4. A.S. 2121350, А61К 31/715. "A method for producing an alpha-fetoprotein preparation." / V.V. Starikov, S.Yu. Rodionov, Myakkohodov V.A., Reshetnikov S.S., Pak N.A. (Russia). - No. 96111118/14. Claim 12/01/95. Publ. 12/27/97. Bull. Number 36.

5. Nikitin V.V., Zvyagin I.V. "Freezing and drying of biological products." M .: Kolos, 1971, 344 p.

6. Gold Phil et all. "Physicochemical Approach to the Purification of Human Fetoprotein from the Ascites Fluid of a Hepatoma-bearning" Patient. Cancer research. 1978, - No. 36, - p.6-12.

7. Huse Klaus et all "A novel purification procedure for human fetoproteun by application of immobilized Cibacron Blu F36-A as affinity ligand Clinica ChimicaActa" 133, 1983, 335-340.

Claims (1)

A method of producing alpha-fetoprotein tablets, including mixing with a stabilizer, adding a filler consisting of microcrystalline cellulose and potato starch or lactose, coating the resulting mixture with a shell consisting of acetylphthalyl cellulose in the ratio of ingredients, wt.%: Alpha-fetoprotein - 0.02-0 , 03, stabilizer - 8.57-14.28, MCC - 40-45.7, potato starch or lactose - 40-45.7 and AFC - 1.14-2.85, characterized in that the stabilizer used reopoliglyukin, and alpha-fetoprotein is obtained from abortive, umbilical cord or placental blood, ballast proteins are separated from the blood by centrifugation, the supernatant is fractionated with ammonium sulfate, or sodium sulfate, or ethyl alcohol for 1 h, the solution is centrifuged again at 2500 rpm for 30 minutes, the resulting supernatant is diluted with 0.1-1 , 0 mol / L solution of sodium chloride and 0.01-0.5% solution of triton X-100, carry out the first chromatographic purification of the supernatant on an affinity sorbent using Sepharose CL-4B, alpha-fetoprotein 0.1 M glycine is eluted HCl buffer solution with a pH of 2.5 to reach After zero optical density at a wavelength of 280 nm, then a second chromatographic purification is carried out on an affinity sorbent using CL-4B Sepharose containing immobilized antibodies against human IgG, dialyzed alpha-fetoprotein solution relative to 0.15 mol / L sodium chloride solution, after whereby the target product is diluted with reopoliglucin to a concentration of alpha-fetoprotein 0.9 mg / ml and dextran 10 mg / ml