RU2454221C2 - Method for preparing lyophilised antiviral agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to method, and concerns a method for making a lyophilised antiviral agent consisting of dissolving a peptide in water, mixing the prepared solution with a number of structure-forming substances, sterilising and lyophilising including freezing out and drying. As a peptide, the peptide His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly is used.

EFFECT: invention provides optimising the process enabling preparing the broad-spectrum lyophilised antiviral agent corresponding to the certified pharmacopeial description of the manufacturer.

3 cl, 1 ex, 3 tbl

Description

The claimed invention relates to the pharmaceutical industry, in particular to methods for producing drugs in the form of a lyophilisate with antiviral effect.

A significant number of antiviral drugs are known from the prior art, such as arbidol, amixin (tilorone), ribavirin, acyclovir and others, most of which are available in solid dosage forms, namely tablets and capsules (see, for example, Mashkovsky M.D. Medicines. - 15th ed. M.: New Wave LLC. - 2006).

Currently, the most widely used and used drugs are those made in the form of gelatin capsules, consisting of a partially or completely transparent soft shell, the inner volume of which is filled with granules or powder of a beneficial agent - a drug substance, while the granules release the drug substance immediately or gradually. Known drugs are used to treat cough, colds and / or allergic symptoms (see, for example, application for invention No. 96102843 “Particles enclosed in soft gelatin capsules”, filing date 02/09/1996, publication date 05/20/1998) .

The disadvantages of gelatin capsules include, first of all, high sensitivity to moisture, therefore, to preserve the drug, certain conditions must be met. In addition, gelatin capsules are a favorable medium for the propagation of microorganisms, to prevent exposure to which preservatives, such as nipagin, sorbic acid and others, must be added to the composition of the drug (see, for example, Chueshov V.I., Chernov M.Yu. Industrial technology of drugs. - X .: MTK-Kniga, 2002, - vol. 2; Nikitin EE, Zvyagin IV Freezing and drying of biological preparations. M: Kolos, 1971. - 270 p.).

Based on this, encapsulation is not used for drugs containing peptide molecules. For peptide preparations, lyophilic drying is mainly used.

Lyophilization is a method of soft drying of substances in which the preparation to be dried is frozen and then placed in a vacuum chamber in which sublimation of the solvent takes place.

The advantages of this drying method are: no exposure to high temperatures on the preparation, preservation of the dispersed phase of the preparation, the possibility of using volatile solvents.

The lyophilization method allows you to get dry tissue, drugs, products and the like without losing their structural integrity and biological activity. During lyophilization, most proteins do not undergo denaturation and can persist for a long time with moderate cooling (about 0 ° C).

Lyophilized tissues and preparations, when moistened, restore their original properties (see Daveney T., Gergei Y. Amino acids, peptides and proteins. - M .: Mir, 1976. - 364 p .; Belous AM, Tsvetkov Ts.D. Scientific principles of technology freeze-canning. - Kiev: Science Dumka, 1985. - 208 p .; Mackenzie AP Collaps during freeze-drying - qualitative and quantitative aspects. Freeze-during and advanced food technology / Eds. SAGoldbith, L. Rey, Rothmayr. - London: Academic Press. - P.277-308).

The closest technical to the claimed method for producing a lyophilized drug is a method comprising dissolving 1 part by weight Cetrorelix acetate in 30-15 parts by weight acetic acid, transferring it into water and mixing the solution thus obtained with one or more structuring substances, sterilization and lyophilization (see RF patent for invention No. 2145234 “Peptide-based lyophilisate and method for its preparation”, filing date 02/18/1994 , published on 02/10/2000).

The technical result to which the claimed invention is directed is to optimize a process that allows to obtain a lyophilized antiviral agent of broad action, including for the treatment of diseases caused by herpes viruses, human papillomas, immunodeficiency states characterized by frequent relapses or chronic course of viral infections, for the prevention of cancer diseases of viral etiology and conditions caused by radiation, physical and chemical influences, as well as compliance with its indicators pharmacopoeial article of the enterprise.

This result is achieved in that in a method for producing a lyophilized antiviral agent, consisting of dissolving a peptide in water, mixing the resulting solution with several structuring agents, sterilization and lyophilization, including freezing and drying, according to the invention, sterilization is carried out by double filtration, lyophilization is carried out in a Usifroid lyophilizer , after freezing, the rate of which is 2 ° C / min, the solution is sublimated, while using as a peptide comfort peptide His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly, when mixing the solution, a stabilizing component in the form of aspartic and / or glutamic acid is additionally introduced, and structural agents are used attracts, alanine and / or glycine in the following ratio of components, wt.%:

Peptide His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly 0.19-6.6 Mannitol 66.0-92.0 Alanine and / or Glycine 3.6-13.0 Aspartic Acid and / or Glutamic Acid 3.6-13.0.

The lyophilized antiviral drug obtained by the claimed method is used to treat diseases caused by herpes viruses, human papillomaviruses, immunodeficiency conditions characterized by frequent relapses or a chronic course of viral infections, as well as for the prevention of oncological diseases of viral etiology.

In addition, it is possible the clinical use of the drug in the treatment of conditions caused by radiation, physical and chemical influences.

Technical solutions that coincide with the totality of the essential features of the claimed invention have not been identified, which allows us to conclude that the claimed invention meets such a patentability condition as “novelty”.

The claimed essential features that predetermine the receipt of the specified technical result, do not explicitly follow from the prior art, which allows us to conclude that the claimed invention meets such a patentability condition as "inventive step".

The patentability condition “industrial applicability” is confirmed by the example of a specific application.

An example of a specific implementation method.

The components were used in the following ratio: peptide His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly in an amount of 0.01-10 mg; as structure-forming substances - mannitol in 5-100 mg and / or alanine - 0.2-20 mg (hydrophobic, branched structure); to increase stability, 0.2-20 mg of aspartic acid is introduced into the composition, which neutralizes the strongly basic (positive) charge, increases stability and reduces the disintegration of the agent.

In a glass flask with a capacity of 2 l, approximately 1.5 l of water for injection is placed. On an electronic balance, 1 mg of the peptide is weighed and dissolved in a flask with water for injection. Then weighed 42 mg of mannitol, 2 mg of alanine, 2 mg of aspartic acid. Transferred to a flask with water for injection, all thoroughly alter.

pH of the finished solution 5.9-6.4; density: 1.007-1.012 g / cm ³ at 20 ° C.

The solution is sterilized by double sterilizing filtration through membrane filters under aseptic conditions, while the pore size of the filters is 0.22 μm. 100 ml of the first epaulette discarded. Filters are sterilized with water vapor. The solution after preparation is immediately dosed into ampoules (vials) for injection. Colorless, hydrolytic class 1 ampoules are used, under aseptic conditions, previously sterilized. The filled ampoules for injection are transferred to a freeze dryer and dried at a predetermined temperature. The process of lyophilization of solutions adapted to the volume of the lyophilisate and capacity consists of the following steps: the solution goes through a freezing cycle, then sublimation and drying. Lyophilization is carried out in a Usifroid lyophilizer (France) of the SMH 15, SMJ 100 or SMH 2000 type, the freezing rate being preferably chosen close to −2 ° C. per minute. Drying is carried out using a drying program at a plate temperature increasing from -40 ° C to + 20 ° C. Then the installation is filled with sterile nitrogen, the ampoules are sealed (the bottles in the installation are closed and the plugs are equipped with a flap valve). Visual inspection is carried out for external defects of the primary packaging, control for mechanical inclusions and transparency is carried out using sample dissolution. Ampoules for injection with defects are separated and destroyed.

The finished product is a white powder or porous mass. Storage conditions of the obtained lyophilisate suggest storage in a dry, dark place and out of the reach of children at a temperature of 2-8 ° C.

All components that make up the antiviral agent are approved for use in medical practice and are produced in accordance with the requirements of the State Pharmacopoeia.

On the basis of experimental data and in accordance with the requirements of the Global Fund XI, the norm for the test “Dosing uniformity” was introduced in the FSP.

Based on the fact that the ampoule contains an extremely small amount of material, determining the average weight of the product by weighing is impossible.

In accordance with the State Pharmacopoeia XI, issue 2, p.143 in this case, a test for uniformity of dosage is carried out. The contents of 10 vessels are tested separately. The content of the product in each vessel should not differ from the nominal by more than ± 15%. If in one vessel the deviation exceeds ± 15%, but does not go beyond 75-125%, an additional test of the contents of another 20 vessels is carried out. In this case, in each of the 20 vessels, the deviation of the content of the product from the nominal should not exceed ± 15%.

In addition, a norm for the Pyrogenicity test has been introduced.

Pyrogenicity tests are carried out in accordance with the requirements of the State Pharmacopoeia XI, issue 2, p.183-185. For testing, the solution is heated to 37 ° C and aseptically slowly injected into the ear vein for three rabbits at a dose of 1 mg / kg (0.1 ml / kg) for 30 seconds.

The temperature is measured before the start of the study and three times at intervals of 1 hour after administration. Temperature measurements are carried out using a TPEM-1 electric medical thermometer or similar (permissible basic error from the range of measured temperatures, ± 1%). With the intravenous administration of the prescribed test dose, the drug must withstand the requirements of the State Pharmacopoeia XI, issue 2.

Test for "Stability".

The shelf life of the drug is established experimentally in accordance with the requirements of OST 42-2-72 "Medicines. The procedure for establishing shelf life "for 3-4 years. When choosing the storage temperature and the degree of illumination of the storage location, we proceeded from the physicochemical properties of the active substance (thermal and photolability). The product was stored at a temperature not exceeding -10 ° C in a dry, dark place (conditions of the freezer) for 2.5 years. At the end of the storage period, all indicators corresponded to the pharmacopoeial article of the enterprise.

In the claimed invention, the active substance having antiviral activity is a synthetic linear oligopolypeptide consisting of 13 L-amino acids (His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly Tripeapeptide ) - hereinafter “peptide”. The peptide is an activator of the interferon system and the natural killer system - key components of the immune system.

Conducted experimental preclinical studies, the objects of which were mice, rats, dogs showed that the drug obtained by the described method:

- does not have general toxicity (acute, subchronic and chronic);

- does not have allergenic properties detected in delayed-type hypersensitivity reactions, mast cell degranulation, immune complexes, and conjunctival tests;

- does not affect the reproductive function, in particular, of rats after subcutaneous administration.

In relation to these indicators, preclinical studies were conducted and the following results were obtained.

1. Results of a study of acute toxicity.

To determine the acute toxicity indicators “Composition” in mice and rats, groups of 5 animals were used, which were compared with control groups (similar in number). Animals were randomly assigned to groups. The absence of external signs of disease and the homogeneity of groups by body weight (± 20%) were considered as a criterion for the acceptability of randomization.

Obtained by the claimed method, the drug was administered to white rats and mice of both sexes subcutaneously (s / c) and orally (intragastrically through a probe) in increasing doses according to Litchfield-Wilcoxon. To achieve large doses of the composition, the agent was re-administered to animals at time intervals of 30 minutes for 2–3 hours (up to 6 repeated administrations). Control animals received the same maximum volumes of isotonic sodium chloride solution (6 administrations).

Observations of animals during which clinical symptoms of intoxication were recorded, indicators of general condition were carried out for 14 days. After 14 days, the animals of all experimental groups were subjected to euthanasia (ether overdose) and subjected to pathomorphological examination.

In none of the cases of lethal effects was it possible to achieve even with the introduction of the maximum technically possible doses - 1000 mg / kg for s / c and iv injections. The dose of 1000 mg / kg is 20 thousand times higher than the dose used for humans.

No clinical manifestations of the antiviral effect were observed. The introduction of the composition resulting from the implementation of the method did not cause macroscopic changes in the brain, internal and endocrine organs, edema of the internal organs, etc. On all days of observation, the experimental animals did not differ from the control in general condition and behavior.

Thus, the results of toxicometry, observations of experimental animals in the period after acute use, as well as data of pathomorphological studies make it possible to classify the resulting antiviral agent into class V of practically non-toxic drugs (see H. Hodge et al. Clinical Toxicology of Commercial Products Acute Poisoning, Ed. IV, Baltimore, 1975, 427 p .; KK Sidorov, 1973). The condition of animals that survived acute experiments indicates good tolerance and harmlessness of the composition in doses exceeding the maximum therapeutic for humans by tens of thousands of times.

1.2. Results of a study of subchronic and chronic toxicity.

According to the results of necropsy and histological examination, daily subcutaneous administration of the drug at doses of 0.05.2 and 5 mg / kg for 90 days to rats of both sexes does not cause irritation, inflammation or tissue destruction at the injection sites, and is also not accompanied by the development of dystrophic, destructive, focal sclerotic changes in parenchymal cells and stroma of the internal organs, which indicates good tolerance and harmlessness of the claimed composition.

Daily subcutaneous administration of the composition in different doses for 90 days to dogs of both sexes does not cause dystrophic, destructive, focal sclerotic changes in the parenchymal cells and stroma of the organs under study, and is not accompanied by a local irritating effect or tissue necrosis at the injection site.

1.3. The results of the study of possible allergenic properties.

General anaphylaxis reaction (anaphylactic shock)

The possibility of developing anaphylactic shock after subcutaneous administration of the agent obtained in the described manner to guinea pigs of both sexes at doses of 0.05 and 0.5 mg / kg for 5 days was evaluated. In the control, animals received an equal volume of distilled water.

21 days after the end of the administration of the composition, a resolving dose of the drug was administered intracardially to all experimental and control groups. The reaction was evaluated in Weigle indices. The data obtained are shown in table 1.

Table 1 Assessment of the reaction of general anaphylaxis (Weigle indices) in guinea pigs after administration of an antiviral agent Experimental groups Floor Animal numbers one 2 3 four 5 6 The control M 0 0 0 0 0 0 F 0 0 0 0 0 0 0.05 mg / kg M 0 0 0 0 0 0 F 0 0 0 0 0 0 0.5 mg / kg M 0 0 0 0 0 0 F 0 0 0 0 0 0

Thus, signs of even moderate shock were not detected in any animal from the group of females and from the group of males who received the composition in the studied doses.

Indirect mast cell degranulation reaction

The experiments were performed on guinea pigs in which blood was taken at 10 days after subcutaneous administration of the drug in doses of 0.05 and 0.5 mg / kg to obtain serum. The obtained rat mast cell degranulation indicators in the presence of experimental and control sera, composition or physiological saline in the control are presented in table 2.

table 2 Rat mast cell degranulation in the presence of guinea pig sera treated with an antiviral agent Experimental groups Floor Animal numbers one 2 3 four 5 6 The control M 0.11 0.12 0.11 0.13 0.12 0.13 F 0.12 0.13 0.13 0.13 0.13 0.10 0.05 mg / kg M 0.12 0.12 0.13 0.14 0.12 0.12 F 0.12 0.11 0.11 0.15 0.11 0.10 0.5 mg / kg M 0.11 0.12 0.13 0.12 0.12 0.14 F 0.13 0.13 0.12 0.12 0.10 0.12

Among the obtained indicators of mast cell degranulation (PDTK), in no case did they exceed 0.2, after which this reaction is considered positive (Guidelines for assessing the allergenic properties of pharmacological substances).

In all cases, the indicator was below the specified value, therefore, it is considered negative. The data obtained are presented in table 3.

Table 3 Comparison of mast cell degranulation in experimental and control groups of guinea pigs Experimental groups Floor PDTC, (M ± m) Student t-test Probability (P) one 2 3 four 5 The control M 0.12 ± 0.2 - - F 0.13 ± 0.1 - - 0.05 mg / kg M 0.12 ± 0.2 0.75 > 0.05 F 0.12 ± 0.3 0.15 > 0.05 0.5 mg / kg M 0.12 ± 0.2 0.60 > 0.05 F 0.14 ± 0.2 1.25 > 0.05

Thus, there were no significant differences in the average values of PDTK in the experimental and control groups of female and male guinea pigs receiving the composition at doses of 0.05 and 0.5 mg / kg for a month. These data allow us to conclude that when administered subcutaneously, the composition does not have allergenic properties detected in the indirect mast cell degranulation reaction.

Conjunctival test

A conjunctival test was performed on the 10th day after the end of subcutaneous administration of the drug in doses of 0.05 and 0.5 mg / kg (experimental groups) or an equal volume of solvent for 5 days.

None of the animals from the experimental groups had a reaction to the introduction of the composition. The data obtained indicate that the composition obtained by the claimed method lacks allergenic properties detected in the conjunctival test, and allow us to conclude that the test composition does not have an allergenic effect when administered subcutaneously.

1.4. The results of a study of the effect of composition on reproductive function

During the introduction of the means of reducing the rate of increase in body mass, changes in behavior and death of animals were not observed in any of the studied groups of males or females. The pregnancy index did not significantly differ in the study groups.

An autopsy of the females revealed no increase in the levels of pre- and post-implantation mortality in the experimental groups compared with the corresponding control groups. The females left to give birth did not show any features in the course of childbirth and care for the offspring.

The death rates of newborn rat pups, the ratio of males and females in the litter in the studied groups did not differ. The terms of the detachment of the auricle, the appearance of the primary hairline, the eruption of the incisors were synchronous in the litters of the experimental and control groups. Opening of the eyes in the offspring of males and females receiving the composition occurred almost at the same time as in the control group. The lowering of the testes and the opening of the vagina by the timing also did not differ in the studied groups.

It is known that the active substance - peptide during sterilization is prone to decomposition, to prevent this process, lyophilisate is used. In the finished form, the amount of biologically active substance in the lyophilized solution is about 0.01 to 10 mg. If only the active substance is lyophilized, then an extremely small amount of loose powder is obtained, which is deposited mainly on the walls of the ampoule or vial, and after steam sterilization this small amount of powder is removed from the ampoule or vial.

To prevent the entrainment of the active substance, auxiliary substances, including structure-forming ones, are added to the composition of the drug, due to which stable precipitation forms. As such a structure-forming substance is used, for example, attracts.

Mannitol simultaneously performs two functions, namely, it maintains the solid and rigid structure of the volume of the lyophilizate corresponding to the volume of the lyophilized solution, and also provides adjustment of the isotonicity of the reconstituted solution for injection (see, for example, Hora MS, Rana RK, Smith EW, Liophilized formulations recombinant tumor necrosis factor, Pharm. Res., 9 (1), 33-36 (1992).

It is known from the prior art that the degree of hydrolysis of the tridecapeptide in lyophilized form may increase in the presence of mannitol, and this degree of hydrolysis increases with an increase in the amount of mannitol present in the lyophilisate. This is explained by the fact that crystallization of mannitol during lyophilization changes the distribution of water in the lyophilisate matrix. An increase in the amount of water present in the resulting microenvironment of the active principle favors the hydrolysis of the active principle and decreases its stability (see Herman BD, Sinclair BD, Milton N., Nail SL, The effect of bulking agent on the solid state stability of freeze-dried methylprednisolone sodium succinate, Pharma. Res., 11 (10), 1467-1473 (1994).

In order to avoid excessive hydrolysis, amino acids, for example, alanine, are added, which provides greater hydrophobicity to the lyophilisate and prevents hydrolysis. Alanine in this case serves as an additional structure-forming component and prevents the deterioration of the properties of the lyophilisate during sublimation and drying, allowing the lyophilisate to have a larger specific surface area and, therefore, allows faster drying (see Pikal MJ, Freeze-drying of proteins, Biopharm ., 26-30 October 1990).

It is known from information sources that the presence of glycine in the lyophilisate causes crystallization of the molecules in solution during the freeze phase of the lyophilization process (see Korey DJ, Schwartz JB, Effects of excipiens on the cristallization of pharmaceutical compounds during lyophylization, J. Parenteral Sci. Tech. , 43, 2, 80-83 (1989). Such crystallization of the active principle can improve its stability.

Oligopolypeptides are known to gel (see, for example, Powell MF: Pharmaceutical Resarch, 1258-1263 (8), 1991; Dathe M: Int. J. Peptide Protein Res. 344-349 (36), 1990; Szejtli J: Pharmaceutical Technology International, 16-22, 1991). During sterile filtration, a jelly-like layer remains on the filter, which is why it is impossible to maintain the exact concentration of the substance. To prevent gelation, for example, sodium chloride, aspartic and / or glutamic acid are added. Due to the fact that the total charge of the tridecapeptide is strongly basic (positive), for example, aspartic acid is added to neutralize the charge and, accordingly, increase stability and reduce disintegration.

Claims (3)

1. A method of obtaining a lyophilized antiviral agent, consisting of dissolving a peptide in water, mixing the resulting solution with several structuring agents, sterilization and lyophilization, including freezing and drying, characterized in that sterilization is carried out by double filtration, lyophilization is carried out in a Usifroid lyophilizer, after freezing the rate of which is -2 ° C / min, the solution is sublimated, while the peptide is His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly, etc. and mixing the solution, a stabilizing component is additionally introduced in the form of aspartic and / or glutamic acid, and mannitol, alanine and / or glycine are used as structuring substances in the following ratio of components, wt.%:
Peptide His-Gly-Val-Ser-Gly-Trp-Gly-Gln-His-Gly-Thr-His-Gly 0.19-6.6 Attracts 66.0-92.0 Alanine and / or Glycine 3.6-13.0 Aspartic acid and / or glutamic acid 3.6-13.0 2. A lyophilized antiviral agent obtained by the method according to claim 1, used to treat diseases caused by herpes viruses, human papillomas, immunodeficiency conditions characterized by frequent relapses or chronic course of viral infections, as well as for the prevention of oncological diseases of viral etiology. 3. Lyophilized antiviral agent obtained by the method according to claim 1, used in the treatment of conditions caused by radiation, physical and chemical influences.