DE3723781A1 - MEDICINAL PRODUCTS CONTAINING STABILIZED G-CSF (GRANULOCYTE-COLONY-STIMULATING FACTOR) AND METHOD FOR THE PRODUCTION THEREOF - Google Patents

Abstract

A stable granulocyte colony stimulating factor-containing pharmaceutical preparation contains, in addition to the active agent, at least one substance selected from a pharmaceutically acceptable surfactant, saccharide, protein and high-molecular weight compound.

Description

The invention relates to a G-CSF (granulocyte colony stimu medicating factor). In particular concerns the invention is a stabilized drug in which the active ingredient G-CSF against loss of activity or inactivation by adsorption on the wall of the drug containing vessel or by forming compounds, Polymerization or oxidation of the active ingredient is protected.

A number of infectious diseases are treated with chemotherapy treated. However, it has recently been found that chemotherapy causes serious clinical problems, for example, it leads to the formation of drug-resistant drugs Organisms, to change the causing organisms and it causes strong side effects. To this with the Chemotherapy, in which therapeutically active substances, such as antibiotics and bactericides used are related problems attempts to avoid the prophylactic Abilities of the host to activate an infectious organism Use fabric. This should do the above Problems of chemotherapy can be completely eliminated. One of the various prophylactic abilities of the The host is the phagocytic, bactericidal effect of its leukocytes. It is believed that this is the beginning of a bacterial Infection most affected. That's why it stops one considers it important to have the protective skills against infection of the host by supporting the growth of neutrophil cells and increase their differentiation into mature cells. G-CSF is one of them very usable substance that shows the activities mentioned. G-CSF and a factor-containing drug for preventing infection Diseases is described in EP-A-215 126.

So it is with the chemotherapy currently practiced various inevitable difficulties, and  intensive efforts have been made, a drug to use the prophylactic functions of the Can activate host or the infected person.

As is known, G-CSF can prevent the prophylactic functions of the Activate hosts. In addition, G-CSF shows even larger therapeutic ones Effects in clinical use when combined is used with a fabric that itself prophylactic capabilities of the host activated.

G-CSF is used in very small quantities. Usually is a drug in a dosage of 1 to 7 times per Week and adult administered the 0.1 to 500 µg (preferably contains 5 to 50 µg) of G-CSF. However, G-CSF does Tendency from the wall of his container, for example an injection ampoule or a syringe, adsorbed to will. That is why the active ingredient gets off the wall of his Container, for example an ampoule or a syringe, adsorbed when used for injection, for example as an aqueous Solution that is used. G-CSF therefore either does not develop its full pharmaceutical activity or it is required to use G-CSF in excess so that part can be lost through adsorption.

G-CSF is also unstable and highly sensitive to the environment influences such as temperature, humidity, oxygen and ultraviolet radiation. When exposed to such factors there are physical or chemical changes in G-CSF, for example, compounds, polymerized or oxidized so that it suffers a severe loss of activity. Because of this, it is difficult to be accurate and complete Carrying out therapy by administering very small Ensure G-CSF quantities.

The invention is therefore based on the object of a medicament to provide that contains stabilized G-CSF in which  the active ingredient G-CSF completely against loss of activity is protected.

According to the invention, this object is achieved in that the drug is a pharmaceutically acceptable interface active agent, saccharide, protein or pharmaceutical compatible high molecular weight compound.

The object of the invention is thus a stabilized G-CSF containing drug that contains both G-CSF and at least one pharmaceutically acceptable surfactant Agent, saccharide, protein or a pharmaceutically acceptable contains high molecular compound. Possibly the medicament according to the invention also contains auxiliaries and Additives.

The G-CSF contained in the pharmaceuticals according to the invention is in a manner known per se or as in EP-A-169 566 and EP-A-215 126. For example human G-CSF can be grown either by breeding of a cell strain, such as that of the CNCM under the deposit Create number I-315 or I-483 deposited cell strain, that from tumor cells from patients with oral cancer was obtained, or by expression of a recombinant DNA encoded using a human G-CSF Gene was constructed in a suitable host cell, for example E. coli, C 127 or ovarian cells chinese hamsters.

Any of them can be used in the medicament according to the invention use high purity human G-CSF. Preferred human G-CSF's become a human from the supernatant G-CSF-producing cell culture isolated or are polypeptides or glycoproteins with the biological activity of human G-CSF created by transforming a host with a  recombinant vector were obtained, in which one for one Polypeptide with the biological activity of human G-CSF coding gene was incorporated.

Below are two particularly preferred examples of human G-CSF listed.

The first human G-CSF has the following physically chemical properties:

• 1. Molecular weight: Approximately 19 000 ± 1000 determined by electrophoresis a sodium dodecyl sulfate polyacrylamide gel.
• 2. Isoelectric point: At least one of the three isoelectric points pI = 5.5 ± 0.1, pI = 5.8 ± 0.1 and pI = 6.1 ± 0.1.
• 3. Absorption of ultraviolet light: An absorption maximum is 280 nm and an absorption minimum at 250 nm.
• 4. The amino acid sequence of the 21 N-terminal amino acids is H₂N-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser- Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln-Val-

The second most preferred human G-CSF contains either a polypeptide with the biological activity of human granulocyte stimulating factor that at least has part of the amino acid sequence below, or a glycoprotein other than this polypeptide still has a sugar chain:

(with the proviso that m has the value 0 or 1; and that n has the value 0 or 1).

Details of the manufacturing process of these two G-CSF Shapes can be found, for example, in EP-A-169 566 and See EP-A-215 126.

In another manufacturing process, a G-CSF is being manufactured Cell with a proliferating malignant tumor cell fused and the hybridoma thus obtained if necessary bred in the presence of a mitogen.

The human G-CSF obtained containing solution after cleaning and concentration in frozen state. On the other hand, the solution even after dehydration, for example by Freeze drying, store.

Each of the human G-CSF's obtained in this way can be seen in described the present invention, to drugs  process that ent the G-CSF in a stabilized form hold.

Specific examples of surfactants that are for the production of the stabilized G-CSF according to the invention containing drug are non-ionic limits surfactants with an HLB value of 6 to 18, such as Sor bitan esters of aliphatic acids (e.g. sorbitan monocaprylate, Sorbitan monolaurate or sorbitan monopalmitate), aliphatic Gly cerine esters of aliphatic acids (e.g. glycerol monocaptylate, glycerol monomyristate or glycerol monostearate), polyglycerol ester aliphatic acids (e.g. decaglyceryl monostearate, decagly ceryl distearate or decaglyceryl monolinoleate), polyoxyethylene sorbitan esters of aliphatic acids (e.g. polyoxyethylene sorbi tanmonolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monoplamitate, Polyoxyethylene sorbitan trioleate or polyoxyethylene sorbitan tri stearate), polyoxyethylene sorbitol esters of aliphatic acids (e.g. polyoxyethylene sorbitol tetrastearate or polyoxyethylene sorbitol tetraoleate), poly ethylene glycerol esters of aliphatic acids (e.g. polyoxyethylene glyceryl monostearate), esters of aliphatic acids with polyethylene glycol (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ether (e.g. polyoxyethylene polyoxy propylene glycol ether, polyoxyethylene polyoxypropylene propyl ether or polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ether (e.g. polyoxyethylene nonylphenyl ether), polyoxyethylated castor oil, hardened polyoxyethylated Castor oil (polyoxyethylated hydrogenated castor oil), polyoxyethylated beeswax derivatives (e.g. polyoxyethylated Sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g. polyoxyethylene lanolin) or aliphatic polyoxyethylene acid amides (e.g. polyethylene stearic acid amide); anionic surfactants such as alkyl sulfates with a C₁₀-C₁₈- Alkyl group (e.g. sodium cetyl sulfate, sodium lauryl sulfate or Sodium oleyl sulfate), polyoxyethylene alkyl ether sulfates, in which  the average number of moles of ethylene oxide addition Is 2 to 4 and the alkyl group is 10 to 18 carbon atoms comprises (e.g. polyoxyethylene sodium lauryl sulfate), salts of alkyl sulfosuccinate esters in which the alkyl group 8 has up to 18 carbon atoms (e.g. sodium lauryl sulfo succinate ester); and natural surfactants such as Lecithin, glycerophospholipid, sphingophospholipid (e.g. Sphingomyelin) or the esters of aliphatic acids with Sucrose, in which the aliphatic acid contains 12-18 carbons has atoms of matter. According to the invention, these can be limited surfactants either individually or in a mixture use.

The surfactants listed above will preferably in an amount of 1 to 10,000 parts by weight used per part by weight of G-CSF.

The containing for the preparation of the stabilized G-CSF Medicinal saccharides used are monosaccharides, Oligosaccharides or polysaccharides as well Phosphate esters or their nucleotide derivatives, if they are pharmaceutically acceptable. Typical examples are trivalent and higher sugar alcohols, such as glycerin, Erythritol, arabitol, xylitol, sorbitol or mannitol, sugar acids, such as glucuronic acid, iduronic acid, neuraminic acid, Galacturonic acid, gluconic acid, mannuronic acid, ketoglycolic acid, Ketogalactonic acid or ketogulonic acid, hyaluronic acid or their salts, chondroitin sulfate or its salts, heparin, Inulin, chitin or one of its derivatives; Chitosan or its derivatives, dextrin, dextran with an average Molecular weight of 5 to 150,000, or alginic acid or their Salts. According to the invention, these saccharides can either use individually or in a mixture.

The saccharides listed above are preferred in an amount of 1 to 10,000 parts by weight per part by weight  G-CSF used.

Typical examples of proteins that are used to manufacture of the stabilized drug containing G-CSF, are human serum albumin, human serum globulin, Gelatin, acid-treated gelatin (with an average Molecular weight from 7000 to 100,000), alkali-treated gelatin (with an average molecular weight weight from 7000 to 100,000) or collagen. According to the invention these proteins can either be used individually or in Use mixture.

The proteins listed above are preferably in an amount of 1 to 20,000 parts by weight per Part by weight G-CSF used.

Typical examples of high molecular weight compounds that are for the preparation of the stabilized G-CSF containing Medicinal products are natural polymers such as Hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxy methyl cellulose or hydroxyethyl cellulose; or synthetic Polymers, such as polyethylene glycol (MW = 300 - 6000), Polyvinyl alcohol (MW = 20,000 - 100,000) or polyvinylpyrrolidone (MW = 20,000 - 100,000). These high molecular weight compounds too can be either individually or in accordance with the invention Use combination.

Preferably, the high molecular weight listed above Compounds in an amount of 1 to 20,000 parts by weight used per part by weight of G-CSF.

In addition to the surfactant, saccharide, protein or high molecular compound described above, an amino acid, a sulfurous reducing agent and / or an antioxidant can also be added to the stabilized drug containing G-CSF. Examples of the amino acids which can be used according to the invention are glycine, threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cysteine, cystine and methionine. Examples of the sulfurous reducing agents which can be used according to the invention are N-acetyl cysteine, N-acetyl homocysteine, thioctinic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid or one of its salts, sodium thiosulfate, sodium hydrogen sulfite, glitothesulfite, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium thiol sulfate, sodium sulfate thiol sulfate Reducing agents with a sulfhydryl group, such as a C₁-C₇ thioalkanoic acid. Examples of the antioxidants that can be used in accordance with the invention are erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, dl- α- tocopherol, tocopherol acetate, L-ascorbic acid or one of its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, triamyl gallate, propyl complexate, sodium dendiamate tetraate, chelate diacetate, sodium dicatyl tetraate, chelate diacetate, sodium dicatyl tetraate, chelate diacetate, diamine dextran complex, sodium triamine tetraate, chelate diacetate, sodium diamine complexate, chelate dextran, such as dicylate complexate, chelate diacetate, such as dicylate complexate, chelate dextran, or sodium metaphosphate.

The amino acids listed above, sulfurous reduction agents and antioxidants or their mixtures are preferred wise in an amount of 1 to 10,000 parts by weight used per part by weight of G-CSF.

Furthermore, in the manufacture of the stabilized G-CSF containing drug in a suitable Dosage at least one diluent, a digestion aid, an isotonic agent, an excipient pH modifier, a sedative or a buffer be added.

The stabilized drug containing G-CSF can either for oral or parenteral administration, for example by different types of injection, be formulated. Depending on the mode of administration, the drug used in different dosage forms. Typical Dosage forms are intended for oral administration,  for example as tablets, pills, capsules, granules or suspensions; mainly for intravenous, intramuscular, subcutaneous or intracutaneous injection Solutions, suspensions or freeze-dried preparations; and those intended for transmucosal administration Dosage forms such as rectal suppositories, nasal drugs or vaginal suppository.

According to the invention, the drug containing G-CSF at least one surfactant, saccharide, protein or added a high molecular compound to prevent that the G-CSF from the wall of its vessel or from which is adsorbed into a syringe and to ensure that it is stabilized for a long time at the same time.

The exact mechanism by which the above Substances stabilize the G-CSF or its adsorption prevent has not yet been elucidated. In the presence of a surfactant, the surface could of the hydrophobic G-CSF protein with the surfactant Means be covered. This will solve the G-CSF. Consequently G-CSF, which is only present in traces, is effective before Adsorption on the wall of its container or a syringe protected. A saccharide or a high molecular compound could be a hydrated layer between G-CSF and the ad sorbent surface of the wall of the container or the Form syringe. This also makes the adsorption of the G-CSF effectively prevented. A protein could be around G-CSF Adsorption on the wall of the container or syringe compete. This would make the adsorption of G-CSF effective be inhibited.

The substances mentioned above not only prevent adsorption of the G-CSF but they also support prevention the polymerization of the G-CSF molecules. In the presence of a surfactant, saccharide, protein or one  The individual G-CSF molecules are high molecular weight compounds dispersed in these substances. This creates the interaction sufficiently decreased between the G-CSF molecules. This leads to a sufficient reduction in the probability the formation of compounds or the polymerization. In addition, these substances delay the autooxidation of the G-CSF operating at high temperatures or humidity is increased. They also prevent connections from forming between the G-CSF molecules or their polymerization as a result of auto-oxidation. This effect on the delay auto-oxidation of G-CSF or prevention It is further possible to form compounds or polymers by adding an amino acid, a sulfurous reduction increase with or an antioxidant.

The problems described above can be particularly solved with injection solutions and suspensions, but occur also when formulating G-CSF into other dosage forms, for example tablets. The addition of interfaces active agents, saccharides, proteins or high molecular weight Connections is also effective in the latter case.

By adding at least one surfactant, Saccharide, protein or a high molecular compound G-CSF is strongly stabilized and maintains its activity long periods of time. This is demonstrated in the examples below explained. To achieve the results described the choice of the amount of each of these substances and in particular the selection of the lower limit is critical. The following Quantity ranges are preferred: 1 to 10,000 parts by weight surfactant, 1 to 10,000 parts by weight Saccharide, 1 to 20,000 parts by weight of protein and 1 to 20,000 parts by weight of high molecular weight compound per weight share of G-CSF.  

According to the invention, a surfactant Saccharide, protein and / or a high molecular compound used in a special concentration. This will not just the adsorption of the G-CSF on the wall of its Container or the syringe effectively prevented, but also the stability of the G-CSF in the medicament according to the invention elevated. As a result, it becomes possible to administer a low but very accurate dose of G-CSF to patients guarantee. Since G-CSF is expensive, its economic diminishes Use the manufacturing cost of G-CSF medicines containing them.

In the examples described below, the G-CSF Residual activity using one of the following methods certainly:

(a) Soft agar method with mouse bone marrow cells

0.4 ml horse serum, 0.1 ml sample, 0.1 ml suspension of a Mouse bone marrow cell, for example C3H / He (female), with 0.5 to 1 × 10⁵ nuclear cells and 0.4 ml modified McCoy's 5A culture medium containing 0.75% agar are mixed. The mixture is placed in a plastic tissue culture dish cast with a diameter of 35 mm. The solidified mixture is 5 days at 37 ° C in an atmosphere of 5% CO₂ and 95% air cultivated at 100% humidity. After that the number of colonies that form is determined. A Colony must have at least 50 cells. This becomes calculated the activity. It is assumed that a Unit leads to the formation of a colony.

The modified McCoy's 5A culture used in process (a) medium is produced twice concentrated by dissolving of 12 g McCoy's 5A culture medium (Gibco), 2.55 g MEM Amino acid vitamin medium (Nissui Seiyaku Co., Ltd.), 2.18 g Sodium bicarbonate and 50,000 units of potassium penicillin G  twice in 500 ml of distilled water and subsequent sterile filter through a Millipore filter (0.22 µm).

(b) Reverse phase high performance liquid chromatography

Under the following gradient conditions, the G-CSF Residual activity (injected in an amount corresponding to 1 µg) with a reverse phase C8 column (4.6 mm × 300 mm, 5 µm) and a mixture of n-propanol / trifluoroacetic acid as a mobile Phase determined:

The detection was carried out at a wavelength of 210 mm and the residual G-CSF activity was as follows Formula calculated:

The residual G-CSF determined by this method correlates very well with the results of the measurement after the soft agar Method (a) using mouse bone marrow cells were.

The examples illustrate the invention.  

Example 1

5 µg of G-CSF are used with one of those listed in Table I. Stabilizing agent added. The mixture becomes sterile in a 20 mM buffer solution (containing 100 mM sodium chloride; pH 7.4) dissolved. A drug with 5 µg G-CSF per ml obtained, which is then freeze-dried. The Change in activity of G-CSF over time is measured by method (a). The measurement results are summarized in Table I. The one used in the table Expression "Activity (%)" identifies the G-CSF rest activity in relation to the output unit and is determined by defines the following formula:

Freeze drying was carried out as follows:

The G-CSF solution containing a stabilizing agent becomes transferred to a sterile glass ampoule treated with sulfa, Frozen for 4 hours at at least -50 ° C, and one first drying by heating from -40 ° C to 0 ° C during 48 hours with a simultaneous increase in pressure of 0.03 subjected to 0.1 Torr. The second drying process will by heating from 0 ° C to 20 ° C for 12 hours under simultaneous pressure increase from 0.03 to 0.08 Torr. Then the interior of the ampoule is cleaned with sterile, dried nitrogen gas filled to atmospheric pressure to reach. Then the ampoule with a freezer drying rubber stopper closed and with an aluminum sealed lid.  

Table I

Example 2

10 µg G-CSF are used with one of those listed in Table II Stabilizing agent added. The mixture becomes sterile in a 20 mM phosphate buffer solution (containing 100 mM sodium chloride; pH 7.4 dissolved. It will contain a drug Receive 10 µg G-CSF per ml. The agent becomes sterile in sulfate treated glass ampoules that have been sealed. The change in activity of G-CSF depending on the The time in which the solution contained in the ampoules becomes after measured using the method already used in Example 1. The results are summarized in Table II.  

Table II

Example 3

10 µg G-CSF are with one of those listed in Table III Stabilizing agent added. The mixture is in a 20 mM phosphate buffer solution (containing 100 mM sodium chloride of pH 7.4) sterile and it contains a drug Obtained 10 µg G-CSF / ml. 1 ml of the agent is in a sulfa-treated, silicone-coated glass ampoule transferred and stored at 4 ° C. The effect of the stabilizing agents when changing the G-CSF adsorption is by measurement the residual G-CSF activity in the solution after 0.5, 2 and Evaluated 24 hours. The measurement is made according to the procedure (b) with reverse phase high performance liquid chromatography carried out. The results are in Table III summarized.  

Table III

Claims (13)

1. Medicament containing stabilized G-CSF (granulocyte Colony stimulating factor) and in addition to G-CSF as Active ingredient at least one pharmaceutically acceptable surfactant, saccharide, protein or a pharmaceutically acceptable high molecular compound. 2. Medicament according to claim 1, characterized in that the amount of the surfactant is 1 to 10,000 parts by weight per part by weight of G-CSF. 3. Medicament according to one of claims 1 or 2, characterized in that the surfactant Means a non-ionic, an anionic or is a natural surfactant, wherein the non-ionic surfactant is a sorbitan ester an aliphatic acid, a glycerol ester aliphatic acid,  a polyglycerol ester of an aliphatic acid, a poly oxyethylene sorbitan ester of an aliphatic acid, a Po lyoxyethylene sorbitol ester of an aliphatic acid Polyoxyethylene glycerol ester of an aliphatic acid, an ester of a aliphatic acid with polyethylene glycol, a polyoxyethylene alkyl ether, a polyoxyethylene polyoxypropylene alkyl ether, a polyoxyalkylene alkylphenyl ether, a hardened polyoxyethylated Castor oil, a polyoxyethylated beeswax derivative Polyoxyethylene lanolin derivative or an aliphatic Is polyoxyethylene amide; the anionic surfactant Means an alkyl sulfate, a polyoxyethylene alkyl ether sulfate or is an alkyl sulfosuccinyl ester salt; and the natural surfactants lecithin, glycerin phospholipid, sphingophospholipid or an ester of one is aliphatic acid with sucrose. 4. Medicament according to claim 1, characterized characterized in that the amount of saccharide 1 to 10,000 parts by weight per part by weight of G-CSF. 5. Medicament according to one of claims 1 or 4, characterized in that the Saccharide glycerin, erythritol, arabitol, xylitol, sorbitol, Mannitol, glucuronic acid, induronic acid, galacturonic acid, Neuraminic acid, glyconic acid, mannuronic acid, ketoglycolic acid, Ketogalactonic acid, ketogulonic acid, hyaluronic acid or one their salts, chondroitin sulfate or one of its salts, Heparin, inulin, chitin or one of its derivatives, chitosan or one of its derivatives, dextrin, dextran with one average molecular weight from 5,000 to 150,000; or alginic acid or one of its salts. 6. Medicament according to claim 1, characterized characterized in that the amount of protein 1 to 20,000 Parts by weight per part by weight of G-CSF.   7. Medicament according to one of claims 1 or 6, characterized, that the protein human serum albumin, human Serum globulin, gelatin, acid or alkali treated Average molecular weight gelatin from 7,000 to 100,000 or collagen. 8. Medicament according to claim 1, characterized in that the amount of high molecular weight Connection 1 to 20,000 parts by weight per Part by weight G-CSF. 9. Medicament according to one of claims 1 or 8, characterized in that the high molecular compound hydroxypropyl cellulose, hydroxy methyl cellulose, sodium carboxymethyl cellulose, hydroxy ethyl cellulose, polyethylene glycol with a molecular weight from 300 to 6000, polyvinyl alcohol with one molecule Lar weight from 20,000 to 100,000 or polyvinyl pyrrolidone with a molecular weight of 20,000 to 100,000. 10. Medicament according to one of claims 1 to 9, characterized characterized in that in addition an amino acid, a sulfurous reducing agent or an antioxidant medium is included. 11. Medicament according to claim 10, characterized in that the amount of amino acid, the sulfurous reducing agent and / or the Antioxidant 1 to 10,000 parts by weight per Part by weight G-CSF.   12. Process for producing a stabilized containing G-CSF drug, characterized in that the active ingredient G-CSF with a pharmaceutically acceptable surfactants, a saccharide, a Protein and / or with a pharmaceutically acceptable high molecular compound, optionally with a Amino acid, a sulfurous reducing agent and / or an antioxidant, and optionally with auxiliary and additives. 13. Use of at least one of the compounds according to one of the Claims 3, 5, 7 or 9 in an amount according to one of the Claims 2, 4, 6 or 8 for stabilizing G-CSF in a medicine.