RU2025120C1 - Method of preparing of preparation containing factor g-csf stimulating the growth of granulocyte colonies - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry. SUBSTANCE: for preparing of preparation containing factor stimulating granulocyte colony growth at least one substance is used taken from the group of pharmaceutically acceptable surface-active substance, saccharide, protein and high-molecular compound. EFFECT: enhanced stability. 2 cl, 3 tbl

Description

 The invention relates to the pharmaceutical industry and relates to a method for producing a preparation containing a factor (G-CSF), stimulating the growth of colonies of granulocytes (GFSC).

 The aim of the invention is to increase stability.

 Currently undergoing chemotherapy, there are various unavoidable problems; intensive efforts are being made to use a medicinal substance capable of activating the preventive functions of a host or a subject that is infected.

 FSHC has the ability to activate the prophylactic functions of the host, in addition, it has been found that FSHC exhibits higher therapeutic effects in clinical applications if it is used in combination with a substance that activates the prophylactic capabilities of the host.

 FSKG is used in very small quantities, a pharmaceutical preparation containing 0.1-500 μg (preferably 5-50) FSKG is usually administered with a dosage intensity of 1-7 times a week per adult patient. However, FSKG has a tendency to adsorb onto the wall of the container, such as an injection ampoule or syringe. Therefore, if the medicine is used as an injection in the form of an aqueous solution, it will be adsorbed on the wall of the container, for example, an ampoule or syringe. This leads to the inability of FSKG to fully show activity as a pharmaceutical agent or entails the introduction of FSKG in more than the required amount, taking into account its possible loss due to adsorption.

 In addition, FSKG is labile and extremely susceptible to environmental factors, such as temperature, humidity, oxygen and UV rays. Under the influence of such factors, FSKG undergoes physical and chemical changes: co-generation, polymerization and oxidation - and suffers large losses in activity. These phenomena make it difficult to fully implement the therapeutic effect by administering a very small amount of FSKH in a strict manner. Therefore, there is a need to develop a sustainable pharmaceutical product FSKG, which is fully protected from the loss of activity of its effective component. This is the main objective of the invention, which offers a sustainable pharmaceutical product FSKG.

 The method provides intensive research in order to increase the stability of FSKG. The goal can be effectively achieved by the addition of a pharmaceutically acceptable surfactant, saccharide, protein, or high molecular weight compound.

 Therefore, a stable pharmaceutical preparation containing FSKG is characterized by a content of FSKG and at least one substance selected from the group of pharmaceutically acceptable surfactants, saccharide, protein or high molecular weight compounds.

 FSKG contained in the pharmaceutical preparation of the present invention can be obtained by any of the known methods described in Japanese patents NN 153273 (1984), 269455 (1985), 270838 (1985) and 270839 (1985).

 The resulting solution containing FSKG can be stored in a frozen state after further purification and concentration using any known method. Alternatively, the solution can be stored after its dehydration by methods such as, for example, lyophilization.

 All FSKG thus obtained can be processed to obtain stable FSKG-containing pharmaceuticals.

Typical examples of a surfactant that is used to produce a stable FSHC-containing pharmaceutical preparation of the present invention are listed below. Nonionic surfactants with H1B 6-18, such as aliphatic acid sorbitan esters (e.g. sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate), aliphatic acid glycerol esters (e.g. glycerol monocaprylate, glycerol mono-amineric acid glycerol monomers, , decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate), polyoxyethylene sorbitan aliphatic acid esters (e.g. polyoxyethylene sorbitan monolaurate, polioaksietilensorbitanmonooleat, polyoxyethylene sorbitan monostearate, polioksietilensorbitanmonopalminat, and polioksietilensorbitantristearat polyoxyethylene sorbitan trioleate), polyoxyethylene aliphatic acid esters (e.g., polioksietilensorbittetrastearat and polioksietilensorbittetraoleat) polioetilenglitserinovye complex aliphatic acid esters (e.g., polioksietilenglitserilmonostearat), polyethylene glycol aliphatic acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polioksietilenlaurinovy ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., ether polioksietilenpolioksipropilenglikolevy, polioksietilenpolioksipropilenpropilovy polioksietilenpolioksipropilentsetilo- ether and polyglycol ethers), polyoxyethylene alkyl phenol ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxyethylated castor oil, hardened polioksietilirovanoe castor oil (polyoxyethyl hydrogenated castor oil), polyoxyethylated beeswax derivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g. polyoxyethylene lanolin) and polyoxyethylene aliphatic amides (e.g. stearic acid polyethylene amide). Anionic surfactants such as alkyl sulfuric acid salts having a C ₁₀ -C ₁₈ alkyl group (e.g. sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate), sulfuric acid salts of polyoxyethylene alkyl ether, where the average molar number of addition of ethylene oxide is 2-4 and the alkyl group contains 10-18 carbon atoms (e.g. sodium polyoxyethylene lauryl sulfate), salts of alkyl sulfosuccinate esters, where the alkyl group contains 8-18 carbon atoms (e.g. sodium lauryl ether th). As well as natural surfactants such as lecithin, glycerophospholipid, sphingophospholipid (for example, ofingomyelin) and sucrose aliphatic esters, where the aliphatic acid has 12-18 carbon atoms. These surfactants, of course, can be used either alone or in a mixture.

 The surfactants given above are preferably used in amounts of 1-1000 parts by weight. by weight FSKG.

 The saccharide used in the preparation of the stable FSHC-containing pharmaceutical preparation of the present invention can be selected from monosaccharides, oligosaccharides and polysaccharides, as well as phosphate esters and their nucleotide derivatives, provided that they are pharmaceutically acceptable. Typical examples are given below: triatomic and higher saccharide alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol, acidic sugars, such as glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, gluconic acid, mannuronic acid, ketoglycolic acid , ketogalactonic acid, ketogulonic acid, hyaluronic acid and its salts, chondroitin sulfate and its salts, heparin, inulin, chitin and its derivatives, chitosan and its derivatives, dextrin, dextran with medium wt. 5000-150000, alginic acid and its salts. All of these saccharides can be used advantageously either alone or in a mixture.

 The saccharides described above are preferably used in amounts of 1 to 10,000 parts by weight. by weight FSKG.

 Typical examples of the protein used in the preparation of the stable FSHC-containing pharmaceutical preparation of the present invention are human serum albumin, human serum globulin, gelatin, gelatin treated with acid (average mol. M. 7000-100000), gelatin, treated with alkali (average mol. M. 7000-100000), as well as collagen. These proteins can be used both independently and in a mixture.

 These proteins are preferably used in amounts of 1-20000 parts by weight. by weight FSKG. Typical examples of the high molecular weight compound used in the preparation of the stable FSHC-containing pharmaceutical preparation of the present invention include natural polymers such as hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose and hydroxyethyl cellulose, as well as synthetic polymers such as polyethylene glycol (mol. 300-6000), watering alcohol (mol. m. 20000-100000) and polyvinylpyrrolidone (mol. m. 20000-100000). These macromolecular compounds can be used both independently and in combination.

 The high molecular weight compounds described above are preferably used in amounts of 1-20000 parts by weight. by weight FSKG.

 In order to formulate a stable FSHC-containing pharmaceutical preparation of the present invention in a suitable dosage form, one or more of the following agents may be administered: diluent, dissolving excipient, isotonic substance, inert filler, pH modifier, emollient and buffer.

 The stabilized FSKG pharmaceutical preparation of the present invention can be prepared either for oral administration or for oral administration, for example, by injection administered in various ways; a wide variety of dosage forms depends on the specific mode of administration. Typical dosage forms: for oral administration, such as tablets, pills, capsules, granules and suspensions, solutions, suspensions and lyophilized preparations intended for internal injection, and for transmucosal administration (through the mucous membrane), such as rectal suppositories, nasal drugs and vaginal suppositories.

 In accordance with the present invention, at least one substance selected from the group consisting of a surfactant, saccharide, protein or high molecular weight compound is added to an FSKG-containing pharmaceutical preparation so that it is protected from adsorption on the wall of its container or syringe and at the same time remained stable for a long period of time.

 A similar mechanism by which these substances stabilize FSKG or prevent it from adsorption is not yet known. In the presence of a surfactant, the surface of the FSKG, which is a hydrophobic protein, would be protected by the surfactant from being dissolved, so that the FSKG present in trace amounts is effectively protected from adsorption on the wall of its container or syringe. A saccharide or hydrophilic macromolecular compound could form a hydrated layer between FSKG and the adsorbing wall of its container or syringe, thereby effectively preventing the adsorption of FSKG. The protein could compete with FSKG due to adsorption on the wall of its container or syringe, effectively inhibiting the adsorption of FSKG.

 In addition to preventing adsorption of FSKG, these substances could also help prevent the association or polymerization of FSKG molecules. In the presence of a surfactant, saccharide, protein or high molecular weight compound, individual FSKG molecules disperse in these substances and the interaction between FSKG molecules is significantly reduced, which significantly reduces the likelihood of their association or polymerization. In addition, these substances could inhibit the self-oxidation of FSHC, which accelerates at high temperature or humidity, or prevent the association or polymerization of FSHC as a result of its self-oxidation. These effects of inhibiting the self-oxidation of FSKG or preventing it from association or polymerization could be further enhanced by the addition of an amino acid, sulfur reducing agent or antioxidant.

 The described problems deserve special attention when using solutions for injection and suspensions, however, they also occur during the processes of compiling FSKG in any dosage forms, for example tablets. The addition of surfactants, proteins, saccharides or high molecular weight compounds is also effective in this latter case.

 By adding at least one substance selected from the group consisting of a surfactant, saccharide, protein or high molecular weight compound, FSKH is highly stabilized and retains its activity over an extended period of time. To achieve these results, the amount of each of these substances, in particular its lower limit, is crucial, the following intervals are desirable: 1-10000 wt. including surfactants, 1-10000 parts by weight saccharide, 1-20000 parts by weight protein and 1-20000 parts by weight macromolecular compounds per 1 wt.h. FSKG.

 In accordance with the invention, the surfactant, saccharide, protein and / or macromolecular compound is used in a precisely defined concentration, which is effective not only to prevent FSHC from adsorption on the wall of the container or syringe, but also to increase the stability of the FSHC-containing pharmaceutical preparation. As a result, it becomes possible to guarantee the introduction of a small, but extremely accurate dose of FSKG to patients, since FSKG is an expensive drug, its effective use will lead to lower costs for producing FSKG-containing pharmaceuticals.

 The following examples are provided to further illustrate the present invention. In these examples, the residual activity of FSKH was determined by one of the following methods.

(and). Mice bone marrow soft agar method
Horse serum (0.4 ml), 0.1 ml of sample, 0.1 ml of suspension of bone marrow cells of mice (female) SZN (He) 0.5-1 x 10 ⁵ nuclear cells and 0.4 ml of the modified McCoy'S culture solution 5A containing 0.75% of agar were mixed, poured into a plastic dish for tissue culture (diameter: 35 mm), coagulated, and cultured for 5 days at ³⁷ ° C in a 5% CO ₂ and 95% air at 100% humidity. Count the number of colonies formed (one colony consists of at least 50 cells), the activity is determined from the calculation of one unit corresponds to the activity for the formation of one colony.

 The modified McCoy'S 5A culture solution used in method (a) is prepared using the following procedures.

 Modified culture solution McCoy's 5A (double concentration).

 12 g of a culture solution of McCoy's 5A (Gibco), 2.55 g of MEM medium amino acid-vitamin (Nissui Seiyoku Co., Ltd), 2.18 g of sodium bicarbonate and 50,000 units. potassium penicillin G is dissolved twice in 500 ml of distilled water, the solution is aseptically filtered through a millipore filter (0.22 μm).

 (b) High-resolution liquid chromatography with reversed phases.

емя Раств

ель (А) Раст

тель (В) Г

Растворитель (А): 30%-й н-пропал и 0,1%-я трифторуксусная кислота. Растворитель (В): 60%-й н-пропанол и 0,1%-я трифторуксусная кислота. Определение проводят при длине волны 210 нм, процентное содержание остаточной активности ФСКГ вычисляют по следующей формуле:

=

100
Остаточное количество ФСКГ, определенное данным методом, очень хорошо согласуется с результатом, полученным при измерении методом мягкого агара (а) с использованием клеток костного мозга мышей.Using a C8 column with reversed phases (4.6 mm x 300 mm, 5 μm) and a mixture of n-propanol / trifluoroacetic acid as the mobile phase, the residual activity of FSKG (introduced in an amount equivalent to 1 μg) was determined under the following gradient conditions.

I am a Solution

spruce (A) Rust

tel (V) D

Solvent (A): 30% n-disappeared and 0.1% trifluoroacetic acid. Solvent (B): 60% n-propanol and 0.1% trifluoroacetic acid. The determination is carried out at a wavelength of 210 nm, the percentage of residual activity of FSKG is calculated by the following formula:

=

100
The residual amount of FSKG, determined by this method, is very well consistent with the result obtained when measured by the soft agar method (a) using mouse bone marrow cells.

× 100 .PRI me R 1. To 5 μg FSKG add one of the stabilizers shown in the table. 1, the mixture was septically dissolved in a 20 mM buffer solution containing 100 mM sodium chloride, pH 7.4, to obtain a pharmaceutical preparation containing 5 μg FSKG per 1 ml, which was then lyophilized. The time-dependent change in the activity of FSKG is measured by method (a), the results are shown in table. 1. Activity (%) means the residual activity of FSKG relative to the original unit and is determined by the following formula:
Activity (%) =

× 100.

Lyophilization is carried out as follows. G-CSF solution containing a stabilizer, placed in a sterile sulfaobrabotannuyu glass vial, frozen at ^-40 ° C or below for 4 hours, subjected to primary drying by heating from -40 ^{° C to} 0 ° C for 48 h with increasing pressure from 0.03 up to 0.1 mm RT. Art. Then subjected to secondary drying by heating from 0 ^to 20 ° C for 12 h with increasing pressure from 0.03 to 0.08 mm Hg, after which the interior tube is filled sterialnym dry nitrogen gas for the atmospheric pressure and freeze-dried stoppered test tube rubber stopper, and then sealed with an aluminum cap.

 PRI me R 2. To 10 μg FSKG add one of the stabilizers shown in the table. 2, the mixture is aseptically dissolved in 20 mM phosphate buffer solution containing 100 mM sodium chloride, pH 7.4, to obtain a pharmaceutical preparation containing 10 μg FSKG per 1 ml. The drug is aseptically loaded into a sulfonated glass tube and sealed to obtain a FSKG solution. The time-dependent change in the activity of FSKG in this solution is measured using the same method used in example 1, and the results are shown in table. 2.

PRI me R 3. To 10 μg add one of the stabilizers (see table. 3), the mixture is aseptically dissolved in a 20 mm solution of phosphate buffer containing 100 mm sodium chloride, pH 7.4, to obtain a pharmaceutical preparation containing 10 μg FSKG per 1 ml. One milliliter of the preparation was charged into sulfaobrabotannuyu, silicon coated glass vial and left at 4 ° ^C. The efficacy of each stabilizer in preventing G-CSF adsorption was calculated by measuring the residual activity of G-CSF in the solution after 0.5, 2 and 24 hours. The measurement is carried out by the method (b) from using reverse phase high performance liquid chromatography. The results are shown in table. 3.

Claims (2)

1. METHOD FOR PRODUCING A PRODUCT CONTAINING A FACTOR / G-CSF / STIMULATING THE GROWTH OF GRANULOCYTIC COLONIES, characterized in that, in order to increase stability, G-CSF is mixed at a temperature of from 1 to 30 ^° C with at least one substance selected from a group comprising a pharmaceutically acceptable surfactant, a saccharide, a protein and a high molecular weight compound in the quantities listed in the order listed: 1 to 10,000 parts by weight, 1 to 10,000 parts by weight, 1 to 20,000 parts by weight and 1 to 20,000 wt. hours for 1 hour G - CSF, then the mixture is dissolved in a buffer solution, the aforementioned factor G - CSF has the following physicochemical properties: a) molecular weight of about 19000 ± 1000, measured by electrophoresis through sodium dodecyl sulfate-polyacrylamide gel; b) isoelectric point: has at least one of three isoelectric points pI = 5.5 ± 0.1; pI = 5.8 ± 0.1 and pI = 6.1 ± 0.1; c) ultraviolet absorption: has a maximum absorption at 280 nm and a minimum absorption at 250 nm. 2. The method according to claim 1, characterized in that they take the amino acid sequence of 21 residues from the N terminal: H ₂ N - ThZ - Pro - Leu-Glu - Pzo - Ala - Ser - Ser - Leu - Fro - Zeh - Ser - Phe - Leu - Leu - Lys - Cys - Leu - Zlu - Zbh - Val -, the surfactant is selected from the group consisting of sorbitan and aliphatic acid ester, aliphatic acid and polyoxyethylene sorbitan ester, polyoxyethylene polyoxypropylene alkyl ether, cured polyoxyethylene cured oil, alkyl sulfate salt and lecithin, a saccharide selected from the group consisting of glycerin, xyl um, mannitol, glucuronic acid, nemaric acid, ketoglycolic acid, heparin, chitin, chitosan, dextrin and alginic acid, the protein is selected from the group consisting of human serum albumin, human serum globulin treated with acid or alkali, gelatin and collagen, a high molecular weight compound a group comprising hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol and polyvinyl pyrrolidone; the buffer solution is selected from the group consisting of phosphate buffer, acetate buffer and citrate phosphate buffer.

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