DE3723781C2 - Use of polyoxyethylene sorbitan esters of an aliphatic acid to stabilize G-CSF (granulocyte colony stimulating factor) - Google Patents

Description

The invention relates to the use of polyoxyethylene sorbitan esters of an aliphatic acid for stabilization of G-CSF (Granulocyte Colony Stimulating Factor). In particular, the invention relates to the use of Polyoxyethylene sorbitan esters of an aliphatic acid Stabilization of drugs in which the active ingredient G-CSF against loss of activity or inactivity tion 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 caused by Chemothera pie 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 anti biotics and bactericides are used, related problems to avoid me, one tried the prophylactic Abilities of the host of an infectious organism aktivie material to use. This should ge mentioned problems of chemotherapy completely eliminated the. One of the various prophylactic abilities of the The host is the phagocytic, bactericidal effect of its leu cozy. It is believed that this marks the beginning of a bak most affected. That's why it stops it is considered important to protect against infection capabilities of the host by supporting the growth of neutrophil cells and to 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 infectious diseases is described in EP 215 126 A1.  

So there are chemotherapy currently practiced associated with inevitable difficulties, and intensive efforts have been made to find a medicine use the substance that has the prophylactic functions of 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 greater therapeutics effects in clinical use if it is in Com combination with a substance that is itself the 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 (before preferably 5 to 50 µg) G-CSF contains. However, G-CSF does Tendency from the wall of his container, for example an injection ampoule or a syringe, adsorbed to become. Therefore, the active ingredient from the wall of his Be ratio, 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 tel that contains stabilized G-CSF in which  the active ingredient G-CSF completely against loss of activity protects.

According to the invention this object is achieved in that the drug is a pharmaceutically acceptable one Polyoxyethylene sorbitan ester of an aliphatic acid adds.

The invention thus relates to the use of Polyoxyethylene sorbitan esters of an aliphatic acid Stabilization of a drug containing G-CSF. The drug may also contain auxiliary and Additives.

The G-CSF contained in the drugs is in a manner known per se or as in EP-169 566 A1 and EP-215 126 A1. At for example, human G-CSF can be grown either by breeding development of a cell strain, like the one behind the CNCM accession number I-315 or I-483 deposited cell strain make up of tumor cells from oral cavity patients 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 can be used in the drug use high purity human G-CSF. Preferred human G-CSF's become human from the supernatant G-CSF-producing cell culture isolated or are polypeptides or glycoproteins with human biological activity G-CSF, which is achieved by transforming a host with a  recombinant vector were obtained, in which one for a Po lypeptide 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 physico-chemical properties:

• 1. Molecular weight:
  Approximately 19,000 ± 1000 determined by electrophoresis through 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 is 250 nm.
• 4. The amino acid sequence of the 21 N-terminal amino acids is
  

The second particularly preferred human G-CSF contains either a polypeptide with the biological activity of the human granulocyte-stimulating factor, which has at least a part of the following amino acid sequence, or a glycoprotein, which besides this polypeptide also has a sugar chain:

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

Details of the manufacturing process of these two G-CSF Shapes can be found, for example, in EP-169 566 A1 and See EP-215 126 A1.

In another manufacturing process, a G-CSF is manufactured lenticular cell with a proliferating malignant tumor cell fused and the hybridoma obtained thereby optionally bred in the presence of a mitogen.

The human G-CSF obtained containing solution can be frozen after cleaning and concentration in Condition. On the other hand, the solution even after dehydration, for example by Ge freeze drying, store.

Each of the human G-CSF's obtained in this way can be described the present invention, to medicinal white process that contain the G-CSF in a stabilized form ten.  

Specific examples of polyoxyethylene sorbitan esters aliphatic acids, which are used to produce the stabilized G-CSF containing drug are Polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene lensorbitan monopalmitate, polyoxyethylene sorbitan trioleate or Polyoxyethylene sorbitan tristearate. Leave according to the invention these esters are used either individually or in a mixture.

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

To produce the stabilized G-CSF containing Medicament can be used as further components such as saccharides Mannitol can be used.

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

As an additional component that is used to manufacture the stabilized drug containing G-CSF is suitable, can use proteins like human serum albumin become.

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

In addition, 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 that 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 sulfide, glitothesulfothiolothiolothiole sulfate, sodium thiol sulfate, sodium thiol sulfate Reductants with a sulfhydryl group, such as a C ₁ -C ₇ thioalkanoic acid. Examples of the antioxidant agents which can be used according to the invention are erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, dl-α-tocopherol, tocopherol acetate, L-ascorbic acid or one of its salts, L-ascorbic acid palate, L-ascorbic acid stearate, triamyl gallate, diacetate diacetate (S) -datylate tolate, sodium dicylate complexate, sodium dicatylate tateate, sodium dicatylate tetracate, sodium trylate complexate, sodium tri-ethylate tateate, sodium tri-ethylate-tetra-acetate, sodium tri-ethylate-tetra-tetra-ethyl acetate, or sodium metaphosphate.

The amino acids listed above, sulfurous reduc tion agent and antioxidant or the mixtures are before preferably in an amount of 1 to 10,000 parts by weight used per part by weight of G-CSF.

Furthermore, ent can be used in the production of the stabilized G-CSF holding drug in a suitable dosing at least one diluent, one Auf closing aid, an isotonic agent, an excipient pH modifier, a sedative or a buffer be added.

The stabilized drug containing G-CSF can ent neither for oral nor for parenteral administration following, for example, by different types of injection, be formulated. Depending on the mode of administration, the drug used in different dosage forms. Ty Dosage forms are preferred for oral administration  see, for example as tablets, pills, capsules, Gra nulate or suspensions; mainly for intravenous, intramuscular, subcutaneous or intracutaneous injection seen solutions, suspensions or freeze-dried pre ready; 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 is used Polyoxyethylene sorbitan ester of an aliphatic acid as surfactant added to prevent the G-CSF from the wall of his vessel or from one Syringe is adsorbed, and to ensure that it is simultaneously stabilized over the long term.

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 the surfactant, the upper surface of the hydrophobic G-CSF protein with the interfaces active funds. 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 compound could be a hydrated layer between G-CSF and the ad sorbent surface of the wall of the container or 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 to be fully inhibited.

The above substances not only prevent the ad sorption of the G-CSF but they also support the prevention Polymerization of the G-CSF molecules. In present of surfactant, saccharide and protein are the individual G-CSF molecules  dispersed in these substances. This creates the interaction sufficiently decreased between the G-CSF molecules. This leads to a sufficient reduction in the probability speed of compound formation or polymerization. In addition, these substances delay the autooxidation of the G-CSF operating at high temperatures or humidity is increased. They also prevent the formation of ver bonds between the G-CSF molecules or their polymers sation due to auto-oxidation. This effect on ver delay in auto-oxidation of G-CSF or prevention It is further possible to form compounds or polymers by adding an amino acid, a sulphurous reduc or an antioxidant.

The problems described above can be particularly solved with injection solutions and suspensions but also when formulating G-CSF to others Dosage forms, for example tablets. The Add polyoxyethylene sorbitan ester of an aliphatic Acid as a surfactant, alone or with the additional components, saccharides and / or proteins but also effective in the latter case.

By adding at least one Polyoxyethylene sorbitan ester of an aliphatic acid as surfactant, alone or with the additional components, saccharides and / or protein G-CSF strongly stabilizes 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, and 1 to 20,000 parts by weight protein per Weight fraction G-CSF.  

According to the invention, a polyoxyethylene sorbitan ester is one aliphatic acid as a surfactant, alone or with the additional components, saccharide and / or protein 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 medium increased. 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 economy is reduced economic use the manufacturing costs for G-CSF medicines containing them.

In the examples described below, the G-CSF Be residual activity using one of the following methods Right:

(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 × 10 ⁵ nuclear cells and 0.4 ml modified McCoy's 5A culture medium containing 0 , 75% agar are mixed. The mixture is poured into a plastic tissue culture dish with a diameter of 35 mm. The solidified mixture is cultivated for 5 days at 37 ° C in an atmosphere of 5% CO ₂ and 95% air at 100% humidity. The number of colonies that form is then determined. A colony must have at least 50 cells. The activity is calculated from this. It is assumed that a unit leads to the formation of a colony.

The modified McCoy's 5A Kul used in process (a) turmedium is produced in two concentrated ways by Auf dissolve 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 Ste Filtration 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 a quantity corresponding to 1 μg) was carried out using a reverse phase C8 column (4.6 mm × 300 mm, 5 μm) and a mixture of n-propanol / trifluoroacetic acid mobile phase determines:

The detection was carried out at a wavelength of 210 nm and the residual G-CSF activity was calculated using the following formula:

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

The examples illustrate the invention.  

example 1

5 µg of G-CSF are mixed with one of the stabilizing agents listed in Table I. The mixture is sterile dissolved in a 20 mmol / l buffer solution (containing 100 mmol / l sodium chloride; pH 7.4). A drug with 5 µg G-CSF per ml is obtained, which is then freeze-dried. The change in activity of G-CSF with time is measured by method (a). The measurement results are summarized in Table I. The expression "activity (%)" used in the table indicates the residual G-CSF activity in relation to the starting unit and is defined by the following formula:

Freeze drying was carried out as follows:
The G-CSF solution containing a stabilizing agent is transferred into a sterile sulfate-treated glass ampoule, frozen for 4 hours at at least -50 ° C., and a first drying by heating from -40 ° C. to 0 ° C. for 48 hours and simultaneously Pressure rose from approximately 4.00 to 13.33 Pa. The second drying process is carried out by heating from 0 ° C to 20 ° C for 12 hours while increasing the pressure from approx. 4.00 to 10.67 Pa. The interior of the ampoule is then filled with sterile, dried nitrogen gas in order to reach atmospheric pressure. The vial is then closed with a freeze-drying rubber stopper and sealed with an aluminum 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 mmol / l phosphate buffer solution (containing 100 mmol / l sodium chloride; pH 7.4 dissolved. It contains a drug Receive 10 µg G-CSF per ml. The agent is sterile in sul filled glass ampoules that have been sealed. The activity change 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 used with one of those listed in Table III Stabilizing agent added. The mixture is in a 20 mmol / l phosphate buffer solution (containing 100 mmol / l sodium chloride of pH 7.4) sterile and a drug is ent maintaining 10 µg G-CSF / ml. 1 ml of the agent is in a sulfate-treated, silicone-coated glass ampoule leads and stored at 4 ° C. The effect of stabilization medium when changing the G-CSF adsorption is measured solution of G-CSF residual 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 chromato graphic done. The results are shown in Table III summarized.

Table III

Claims (3)

1. Use of polyoxyethylene sorbitan esters aliphatic acid to stabilize Granulocyte colony stimulating factor. 2. Use according to claim 1, wherein the Polyoxyethylene sorbitan esters the monooleate or laureate is. 3. Use according to claim 2, with the additional Components of human serum albumin and mannitol.