RU2414237C2 - Sovialem albumin modification as method for stability enhancement for preparing pharmaceutical biological preparation of albimun in complex with gentamycin or stimaden - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a method for albumin stabilisation for preparing based pharmaceutical biological preparations with additional antibiotic (gentamycin) or immunomodulating (stimadene) with mechanisms of action by sovialem protein modification.

EFFECT: invention provides higher stability of copolymer modified forms of albumin to physical (60°C, 10 hours) and chemical (gentamycin, stimadene) actions with an evident protein- denaturing effect.

2 cl, 1 ex, 1 dwg, 5 tbl

Description

The invention relates to the biological or medical industry, in particular to the production technology of an albumin preparation, in the composition of which there is an antibiotic gentamicin or an immunomodulator stimaden.

In medical practice, albumin has been widely used, including with infectious diseases. Clinically effective are regimens for the combined use of albumin in combination with antibiotics (Sort P., Navasa M., Arroyo V., et al. Effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N. Engl. J . Med. 1999; 341: 403-409; Arroyo V. Review article: hepatorenal syndrome - how to assess response to treatment and nonpharmacological therapy. Aliment. Pharmacol. Ther. 2004; 20 (Suppl. 3): 49-54). They also propose using the biological product albumin as the basis, in which, if necessary, one or another antibiotic is added depending on the course of treatment (Temporary instruction for the use of the drug “Albumin C” No. 13-4-03 / 0688; No. 001517-OP; Temporary instruction for the use of the drug "Albumin N" No. 13-4-03 / 0691; No. 001518-OP). The described algorithm for the manufacture of an albumin biological product with an additional antibiotic effect is carried out immediately before administration in rooms unsuitable for this purpose.

The technological regulations for the production of albumin include the mandatory stage of heat treatment (inactivation of infectious agents) of the semi-finished product at 60 ° C for 10 hours (Rusanov VN, Skobelev LI "Fractionation of plasma proteins in the production of blood products." - M .: Medicine, 1983, p. 10). To preserve the properties of solutions of physiologically active proteins, including albumin, in this heat treatment regimen use sugars: 2% glucose (PDR. 1991. Physicians desk reference. 45th ed. Barnhart, Edward R., Publisher. Medical Economics Company, Inc., Oradell, NJ. p.636), 4, 5% sucrose (“Immunoglobulins.” Collection of scientific papers, edited by V.V. Anastasiev. NN: Publishing House of the Nizhny Novgorod Medical Institute, 1993, p. 70); 3% sucrose (PDR. 1991. Physicians desk reference. 45th ed. Barnhart, Edward R., Publisher. Medical Economics Company, Inc., Oradell, NJ. P. 1528).

However, the presence of sugars in the composition of albumin because of the large volumes and intravenous route of administration limits its use, for example, in diabetes (M.K. Dea et al .: Albumin binding of acylated insulin (NN304) does not deter action to stimulate glucose uptake , Diabetes, 2002, vol. 51, p. 762-769; JFDay et al .: Nonenzymatically glucosylated albumin, The Journal of biological chemistry, 1979, vol. 254, p. 555-597).

The closest technical solution for the production of pharmaceutical biological products, which are clinically used in large volumes, are bioorganic stabilizers that do not cause side complications in patients with diabetes. Known as protein stabilizers in plasma pasteurization: sorbitol, lysir trisodium citrate, arginine (patent RU 93004727 "Composition for stabilizing blood plasma. Method for plasma pasteurization and the use of stabilized plasma in therapy", publ. 1996.10.20). Sodium acetyltryptophanate and sodium caprylate are used as stabilizers for albumin solutions. For a 5% albumin solution, the concentrations of each of these stabilizers are 0.004 mol / L. In a 25% albumin solution, the concentrations of sodium acetyltryptophanate and sodium caprylate are increased 5-fold (PDR. 1991. Physicians desk reference. 45th ed. Barnhart, Edward R., Publisher. Medical Economics Company, Inc., Oradell, NJ. P.598- 599). Bayer Corporation stabilizes the albumin preparation with 0.016 mol / L sodium caprylate and 0.016 mol / L acetyltryptophan. To stabilize domestic albumin preparations, it is recommended to use 0.3% sodium caprylate (FS 42-3543-98 “Albumin solution 5, 10 and 20%”) or add caprylate and N-acetyl-DL-tryptophanate to the preparation depending on the concentration of albumin 80 μmol per 1 g of protein (Rusanov V.M., Levin I. “Blood therapeutics”, Medpraktika Publishing House, Moscow, 2004. 284 s).

The disadvantage of all of the stabilizers of protein solutions listed above is their inability to ensure the production of the substance of albumin in combination with drugs such as gentamicin or stimaden.

A pharmaceutical embodiment based on albumin biologics by adding gentamicin or stimaden is unacceptable due to the instability of the protein solution. Already during short-term storage for 24 hours, visible opalescence, fine suspension, up to a slight precipitate, are observed. Additives of gentamicin or stimaden to semi-finished albumin stabilized using any of the above stabilizers, and their subsequent heat treatment, causes the precipitation of the protein in the precipitate, which becomes poorly soluble. The latter circumstance indicates the denaturing effect of gentamicin and stimaden on the structure and properties of albumin under incubation conditions for 10 hours at 60 ° C.

The objective of the invention is to create a stable form of albumin with the aim of producing on its basis complex preparations with antibiotic (gentamicin) or immunomodulating (stimaden) effects.

The problem is solved by modifying albumin with sovial - a copolymer of N-vinyl pyrrolidone with acrolein diethyl acetal based on the polycondensation of aldehyde groups of sovial and free ε-amino groups of lysine residues in the composition of albumin (EF Panarin, II Gavrilova. Method for producing water-soluble polymers containing aldehyde groups Auth. certificate No. 560425. BI No. 47, 1977; TB Anikina, EF Panarin, OA Mirgorodskaya, BV Moskvichev, II Gavrilova, GV Samsonov, Method for the production of water-soluble physiologically active polymers. view No. 522198. BI No. 27, 1976; N.N. Kutsenko, Z.A. Kuzina, E.F. Panarin, I.I. Gavrilova.Study of the effect of cationic polymers and surfactants on the resistance of bacteria to kanamycin. Antibiotics and medical biotechnology. 1986. No. 6. P. 422-426; TB Tennikova, EF Panarin, OA Mirgorodskaya, GV Samsonov, BV Moskvichev. Immobilization of the proteolytic enzyme terrilitin on a water-soluble polymer matrix.Chem.-Pharmacist.Journal. 1977. T. 11. Number 7. S.80-90). Reactive aldehyde groups are induced by acid hydrolysis of the acrolein unit of sovial. The polycondensation reaction is carried out under conditions of deprotonation of the free ε-amino groups of lysine.

Optimal albumin constructs are obtained in proportions of 3-5 mol sovial per 1 mol albumin. If the incubation medium contains 3-4 mol of sovial per 1 mol of albumin, then the reaction product is subjected to thermal pretreatment. With a five-fold molar excess of sovial, pre-treatment is not required. Then, gentamicin or stimaden is introduced into the solution of modified albumin and the thermal inactivation stage is carried out at 60 ° C for 10 hours in accordance with the regulatory requirements for the production of human and animal albumin biological products.

The described approach was tested on samples of pharmaceutical human and animal biological products (FS 42 0122-04 “Albumin, solution for infusion 5, 10, 20%” or FS 42-3543-98 “Solution of albumin 5, 10 and 20%”, TU 9382- 004-436877-03 "Albuminat", TU 9382-005-436877-03 "Albumin S", TU 9382-006-436877-03 "Albumin N") or samples of their semi-finished products obtained at the stage prior to heat treatment.

It is known that copolymers of vinyl pyrrolidone with crotonic acid are used to synthesize conjugated forms with antibiotics (M.V. Solovsky, N.V. Nikolskaya, V.M. Denisov Gentamicin antibiotic salts with carboxyl-containing copolymers of N-vinylpyrrolidone, Chemical and Pharmaceutical Journal. Volume 34. , No. 11, 2000, p.21-24). Synthetic vinylpyrrolidone-based copolymers are used to screen antigenic determinants of enzymes of bacterial origin in order to reduce their immunogenicity with the parenteral route of administration to humans (B.V. Moskvichev, M.S. Polyak. Immobilized enzymes. M: CDK. 112 s). Conjugated forms of antigens are also known in which an immunomodulatory role is assigned to artificial polymer chains (V.A. Kabanov, R.V. Petrov, PMKhaitov. Journal of the All-Union Chemical Society named after D.I. Mendeleev, 1982, v.24, No. 4 pg. 57-68). However, the use of sovial modified optimal albumin designs to ensure the proper conduct of the regulatory stage of thermal inactivation in the presence of pharmacological substances with a denaturing effect for the production of a complex drug with additional antibiotic (gentamicin) or immunomodulating (stimulant) effects at the present stage of development of the prior art is not known. In addition to the direct stabilizing effect in the presence of bioorganic compounds with a pronounced denaturing effect, the effect of increased thermal stability of optimal copolymer-modified structures is realized in the technical solution of the invention.

The method is as follows.

A solution of sovial in hydrochloric acid is subjected to heat treatment. After cooling, the sovial activated in the above manner is introduced into the albumin solution and the polycondensation reaction is carried out for 2 hours. With a quantitative ratio of 3-4 mol of sovial per 1 mol of albumin, preliminary heat treatment is additionally carried out. Gentamicin or stimaden is added to the solution of sovial modified albumin, followed by the routine heat treatment step.

Examples of specific performance of the method.

A weighed portion of sovial is prepared at a rate of 80-150 mg per 1 ml of 0.01 mol / L hydrochloric acid. A solution of sovial in 0.01 mol / L hydrochloric acid was incubated for 2 hours in a boiling water bath. Then cooled to a temperature of (22 ± 2) ° C. In semi-finished products or preparations of albumin at a protein concentration of 50-100 mg / ml, a solution of sovial is added. The molar ratio should be within 3-5 mol of sovial per 1 mol of albumin. The sovial solution is added gradually to the semi-finished product or albumin preparation under conditions of constant stirring at a speed of 60-70 rpm while adjusting the pH of the incubation medium to (9.5 ± 0.1) using a 0.1 mol / L sodium hydroxide solution. Incubation at pH (9.5 ± 0.1) under the control of a pH meter is carried out for 2 hours with stirring (60-70 rpm), adding, if necessary, 0.1 mol / L sodium hydroxide solution. After 2 hours, the reaction is stopped by adjusting the pH of the incubation medium to (7.0 ± 0.1) with 0.1 mol / L hydrochloric acid. At a ratio of 1 mol of albumin and 3-4 mol of sovial, a preliminary heat treatment stage is carried out at 60 ° C for 10 hours. With a ratio of 1 mol of albumin and 5 mol of sovial, heat treatment is not required.

After the end of the preliminary heat treatment stage, the necessity of which depends on the degree of albumin modification, gentamicin or stimaden is added to the semi-finished product solution. Their maximum possible concentrations are 30 mg / ml gentamicin (for a molar ratio of 5/1), 20 mg / ml gentamicin (for a molar ratio of 3/1), 50 mg / ml of stimaden. This refers to the maximum concentration of pharmacological substances that allow for the stage of the technological process - heat treatment at 60 ° C for 10 hours without the precipitation of albumin. The introduction of an antibiotic or immunomodulator is carried out with stirring. Additional stirring is carried out for another 2 hours. This is followed by the routine stage of thermal inactivation of albumin for 10 hours at 60 ° C. The final preparation is a solution of sovial modified albumin at a molar ratio of copolymer / protein of 3 / 1-5 / 1 and the antibiotic gentamicin (concentration up to 20-30 mg / ml) or immunomodulator stimaden (concentration up to 50 mg / ml). The drawing shows electrophoregrams of samples of copolymer-modified forms of albumin in gradient PAAG (3-25%) obtained by the standard Davis-Laemmli method under native conditions (Laemmli UK Nature, 1970, 227, p.680; Gaal E., Medieshi G. , L. Vereckey, Electrophoresis in the Separation of Biological Macromolecules (Moscow: Mir, 1982, p. 226). The molar ratio of sovial / protein in the tracks: 1-1 / 1, 2-2 / 1, 3-3 / 1, 4-4 / 1, 5-5 / 1, 6-10 / 1, 7-15 / 1, 8-20 / 1, the amount of protein applied - 40 μg per track. Table 1 presents the data on densitometry of electrophoregrams of native albumin and its modified structures. Densitometry of the gels was performed on an ImageScaner Amersham Biosciences)) (United Kingdom) in red light. The area of the entire track was taken as 100%. The fraction of native albumin was calculated as the ratio of the peak area corresponding to the location of native albumin to the total area. The fraction of modified albumin was determined by the difference, minus the fraction of native albumin. Tables 2 and 3 provide data on the effect of modification on the thermal stability of the drug in the presence of gentamicin or stimadenum under the conditions provided for by the regulatory stages of heat treatment. Samples (without preliminary heat treatment) were prepared by adding gentamicin to a concentration of 8 mg / ml and then the regular pasteurization step was carried out (60 ° C, 10 h). Samples with preliminary heat treatment were kept at 60 ° C for 10 hours, then gentamicin or stimaden was added and then subjected to regular pasteurization. The viscosity of the samples was determined on a viscometer type VK-4. From the data in the drawing and in table 1 it follows that with a triple molar excess of sovial, almost all albumin is in a copolymer-modified state. At the same time, the modified forms of albumin, in addition to higher stability under the conditions of protein denaturing effects, acquire increased resistance to proteolytic enzymes (table 4). Hydrolysis was carried out at a molar ratio of enzyme / protein equal to 1/100. Pepsin treatment conditions: 20 min, at pH 2.9 ± 0.05; trypsin: 60 min, pH 8.15 ± 0.05. The fractional composition of hydrolysates was studied by disk electrophoresis in gradient (5-25%) PAAG under dissociating reducing conditions followed by densitometry. Table 5 provides data on the study of the immunochemical properties of native and modified albumin. A direct competitive method in an enzyme-linked immunosorbent assay (ELISA) (using adsorbed immobilized rabbit IgG specific for dog albumin and dog albumin conjugate labeled with horseradish peroxidase) showed a decrease in the level of antigenic determinants in the modified forms of albumin. The percentage of immunochemical similarity (α) of native and modified albumin was calculated as the ratio of concentrations of C _0.5 for specific (native albumin) and modified (3/1 and 5/1) albumin according to the formula

, где

where

With _H0.5 - the concentration of native albumin, leading to 50% inhibition;

With _M0.5 - the concentration of modified albumin (3/1 and 5/1), leading to 50% inhibition.

The calibration dependence for ELISA was prepared from a particularly pure and conformationally native form of dog albumin in concentrations from 0.1 μg / ml to 100 μg / ml (Theory and practice of enzyme-linked immunosorbent assay. / Under the editorship of Egorov AM, Dzantieva BB - M .: High School, 1991 .-- 278 p .; Tijssen R. Practice and theory immunoassays. - Elsevier: Amsterdam New York Oxford, 1985. - 549 p.). The traditional methods of immunoassay used the blood serum of rabbits immunized with native and modified forms of albumin. When determining the titer of antibodies in double immunodiffusion (DID), the antigen (albumin) at a concentration of 1 mg / ml was placed in the central well and immunoserts were triturated in increments (dilution) 2. The presence of cross-reactions was determined by the method of double radial immunodiffusion (Frymel X., Brock I Fundamentals of Immunology. - M., 1986; Antibodies. Methods: Book 1: Translated from English / Edited by D. Catty. - M.: Mir, 1981, pp. 201-207). The results obtained indicate that the screening effect of the modification does not cause the formation of new antigenic determinants capable of inducing antibodies.

Thus, the proposed method for the modification of albumin allows for the production of pharmaceutical biological preparations with an additional antibiotic or immunomodulating mechanism of action in a standard technological process. It is also important that the copolymer-modified forms of albumin proper have increased stability to physical (60 ° C, 10-20 hours) chemical (gentamicin, stimaden) effects, moreover, they are more resistant to protease hydrolysis and do not contain antigenic determinants capable of modifying induce antibodies.

Table 1. The influence of the molar ratio of sovial / protein in the incubation mixture on the degree of modification of albumin (according to electrophoresis under native conditions) Molar ratio
sovial / protein Native share
albumin, X ± S,% Share modified
albumin, X ± S,% 1/1 41.7 ± 12.3 58.3 ± 12.3 2/1 18.0 ± 9.0 82.0 ± 9.0 3/1 6.6 ± 2.9 93.4 ± 2.9 4/1 3.7 ± 2.3 96.3 ± 2.3 5/1 1.9 ± 0.8 98.1 ± 0.8 10/1 0 one hundred 15/1 0 one hundred 20/1 0 one hundred Note: X is the mean value, S is the standard deviation.

Table 2. The effect of the degree of modification on the viscosity and thermal stability of albumin in the presence of gentamicin (8 mg / ml) Sowial / Protein Ratio The relative viscosity of the samples after pasteurization, X ± S Significance of Differences No pre-heat treatment Pre-heat treated 1/1 gel gel - 2/1 9.04 ± 0.96 5.56 ± 0.25 P <0.05 3/1 5.55 ± 1.19 2.53 ± 0.03 P <0.05 4/1 2.87 ± 0.17 2.44 ± 0.09 P <0.05 5/1 3.0 ± 0.3 2.63 ± 0.15 P> 0.05 Note: X is the mean value, S is the standard deviation.

Table 3. Change in albumin viscosity after pasteurization in the presence of various concentrations of stimaden The concentration of stimaden, mg / ml degree of modification, X ± S 0 one 2 3 four 5 2 sediment - - - - - 5-10 - 2.1 ± 0.2 - - - - 10 - gel 3.2 ± 0.3 - - - twenty - - gel - - - 40-50 - - - 4.3 ± 1.5 4.0 ± 0.5 3.4 ± 0.4 one hundred - - - gel gel gel Note: X is the mean value, S is the standard deviation.

Table 4. Fractional composition of hydrolysates of samples of native and copolymer-modified forms of albumin Sowial / Protein Ratio Pepsin hydrolysis,% Trypsin hydrolysis,% Joint pepsin and trypsin hydrolysis,% Mr> 66 kD Mr <66 kD Mr> 66 kD Mr <66 kD Mr> 66 kD Mr <66 kD 0 0 one hundred 27.2 ± 1.1 72.8 ± 1.1 5.98 ± 1.8 94.02 ± 1.8 1/1 13.0 ± 2.1 87.1 ± 2.1 44 ± 5,6 56 ± 5.6 33.7 ± 5.1 66.3 ± 5.1 3/1 35.6 ± 2.7 64.4 ± 2.7 37.7 ± 6.5 62.3 ± 6.5 30.3 ± 2.2 69.7 ± 2.2 5/1 24.1 ± 1.4 75.9 ± 1.4 44.8 ± 1.3 55.2 ± 1.3 29.0 ± 1.7 71.0 ± 1.7 10/1 54.0 ± 3.9 46.0 ± 3.9 65.5 ± 5.8 34.5 ± 5.8 61.8 ± 2.9 38.2 ± 2.9 Note: X is the mean value, S is the standard deviation.

Table 5. The level of similarity of immunogens according to the results of their analysis in the direct competitive ELISA method and the quality of immunoserts induced by native and copolymer modified forms of albumin. Immunogen form The percentage of similarity of immunogens according to the results of their analysis in ELISA (X ± S) The quality of immunogens induced by immunogens By the title of BIT (X ± S) To identify spurs in cross-reactions based on BIT Native albumin one hundred 341 ± 147 Not found The modified form of albumin in a molar ratio of copolymer / protein of -3/1 18.9 ± 4.9 256 ± 128 Not found The modified form of albumin at a molar ratio of copolymer / protein of -5/1 13.2 ± 5.9 149 ± 98 Not found Note X is the mean value, S is the standard deviation

Claims (2)

1. A method for stabilizing albumin for the manufacture of pharmaceutical biologics with additional antibiotic (gentamicin) or immunomodulating (stimaden) mechanisms of action, characterized in that the solution of sovial in hydrochloric acid is subjected to heat treatment, after cooling, the sovial activated in the above manner is introduced into the albumin solution in the calculation of 3- 5 mol of sovial per 1 mol of albumin and carry out the polycondensation reaction for two hours, then gentamicin is added to the solution of sovial modified albumin and and stimaden, followed by heat treatment procedural step. 2. The method according to claim 1, characterized in that the preliminary stage of the heat treatment is carried out at 60 ° C for 10 hours with the ratios: 1 mol of albumin per 3-4 mol of sovial.

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