JP2629000B2 - Stable granulocyte colony stimulating factor-containing preparation - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a granulocyte colony stimulating factor-containing preparation, and particularly to the method of adsorbing on a container wall or losing or inactivating an active ingredient by association, polymerization, oxidation and the like. And a stabilized granulocyte colony stimulating factor-containing preparation.

2. Description of the Related Art Recently, in chemotherapy of various infectious diseases, the emergence of resistant bacteria, the phenomenon of replacement of the causative bacteria, or high side effects have become clinically serious problems. There is a movement to solve the fundamental problem of the above chemotherapy by using a substance which activates the defense function of the host of the infectious bacteria separately from the chemotherapy such as the above. That is, for example, in the early stage of bacterial infection, it is thought that the antiphagic bactericidal action of leukocytes has the strongest effect on the host's defense functions, and it promotes neutrophil proliferation and differentiation / maturation by promoting host infection. It is considered important to enhance defense functions. One of the very important substances having such an action is granulocyte colony stimulating factor (G
-CSF), and an anti-infective agent using the same has already been separately patented by the present applicant (Japanese Patent Application No. 60-23777).
issue).

Problems to be Solved by the Invention As described above, in various chemotherapy, there are various unavoidable problems. For this reason, attempts to use a substance capable of activating the defense function of the infected subject, that is, the host, as a drug are performed. Has been made. G-CSF, of course, has an activity of activating the host's defense function by itself, and its objective is to be exerted even in combination with the above-mentioned drugs in order to more fully exert a clinical therapeutic effect. Has proven to be extremely useful in performing

This G-CSF is used in an extremely small amount, and usually 0.1 to 500 μg (preferably 5 to 50 μg) of G-CSF per adult.
The formulation containing CSF is administered at a rate of 1 to 7 times / week.

However, since this G-CSF exhibits adsorptivity to the walls of, for example, ampoules for injection and syringes, particularly when this drug is used as an injection drug such as an aqueous solution,
Containers such as ampoules, adsorbed on the walls of syringes, etc., are unable to sufficiently exert the pharmaceutical activity of G-CSF, or extra estimate by extrapolating the loss due to such adsorption. It must be added to the drug.

In addition, G-CSF is unstable and susceptible to external factors, causing physical and chemical changes such as association, polymerization or oxidation due to temperature, humidity, oxygen, ultraviolet rays, etc., and has a large activity. Causes a decrease in This makes it difficult to completely perform a therapeutic action that seeks to very precisely administer very small doses of G-CSF.

Therefore, it is necessary to solve such a problem and develop a product capable of sufficiently preventing a decrease in the activity of the active ingredient.
The object of the present invention is in this respect, that is, the stable G-
An object of the present invention is to provide a CSF-containing preparation.

Means for Solving the Problems The present inventors have conducted various studies and studies to improve the stability of the above-mentioned G-CSF-containing preparation, and as a result, it is effective to add a pharmaceutically acceptable protein. Heading that
The present invention has been completed.

That is, the stable G-CSF-containing preparation of the present invention is characterized by containing human recombinant G-CSF and at least one pharmaceutically acceptable protein.

For the human recombinant G-CSF used in the present invention, for example, a recombinant DNA is prepared using a gene encoding human G-CSF, and the recombinant DNA is prepared by using a suitable host cell (eg, Escherichia coli, C127 cell,
Ovarian cells of Chinese hamsters). In particular, a polypeptide or glycoprotein having human G-CSF activity produced by a transformant obtained by transforming a host with a recombinant vector incorporating a gene encoding a polypeptide having human G-CSF activity is preferable. .

Specifically, human G-CS shown in the following (i) and (ii)
F is particularly preferably used.

(I) Human G-CSF having the following physicochemical properties.

Molecular weight: about 19,000 ± 1,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Isoelectric point: pI = 5.5 ± 0.1, pI = 5.8 ± 0.1, pI = 6.1 ± 0.1
Has at least one of the three isoelectric points.

Ultraviolet absorption: has a maximum absorption at 280 nm and a minute value at 250 nm.

The amino acid sequence from the N-terminal to the 21st residue is as follows.

_{H 2 N-Thr-Pro-} Leu-Gly-Pro-Ala-Ser-Ser-Leu-
Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-
Gln-Val- (ii) a polypeptide having human granulocyte colony-stimulating factor activity represented by the following amino acid sequence or a part thereof, or a human G- containing a glycoprotein having the same and a sugar chain portion:
CSF.

(Met) _n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gl
n Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys
Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu (V
al Ser Glu) _m Cys Ala Thr Tyr Lys Leu Cys His Pro G
lu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro
Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu G
ln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu
Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly I
le Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln G
ln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro
Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe G
ln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu
Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg H
is Leu Ala Gln Pro (where m represents 0 or 1 and n represents 0 or 1).

In addition, about the detailed manufacturing method of these G-CSF,
Japanese Patent Application Nos. 59-153273 and 60 filed earlier by the present applicant.
No. -269455, Japanese Patent Application No. 60-269456, Japanese Patent Application No. 60-270838
No., Japanese Patent Application No. 60-270839.

In addition, as another method, it can be obtained by culturing a hybridoma obtained by cell fusion of G-CSF scattered cells and malignant tumor cells capable of self-proliferation in the presence or absence of mitogen.

The human G-CSF-containing solution obtained by these methods can be further purified and concentrated by known means as necessary and then stored frozen, or can be stored by removing water by means such as freeze-drying.

All the human G-CSF thus obtained can be made into a stable G-CSF-containing preparation according to the present invention.

Proteins used to obtain the stable G-CSF-containing preparation of the present invention include human serum albumin, human serum globulin, gelatin, acid-treated gelatin (average molecular weight 7,000 to 100,000), and alkali-treated gelatin (average molecular weight 7,0
00 to 100,000), and collagen as typical examples. These can, of course, be used alone or as a mixture of two or more.

In general, it is preferable to use these proteins in the range of 1 part by weight to 20,000 parts by weight based on 1 part by weight of G-CSF.

The G-CSF-containing preparation of the present invention is a diluent, a solubilizer, an isotonic agent, an excipient, a pH adjuster, depending on the purpose of the preparation.
It may contain a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant and the like. For example, as the sulfur-containing reducing agent, N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, Examples thereof include those having a sulfhydryl group such as a thioalkanoic acid having 1 to 7 carbon atoms.

As antioxidants, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α
-Tocopherol, L-ascorbic acid and its salts,
Chelating agents such as L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate; Can be illustrated.

Alternatively, glycine, cysteine,
Threonine, cystine, tryptophan, methionine,
Amino acids such as lysine, hydroxylysine, histidine and arginine may be added.

The stabilized G-CSF-containing preparation of the present invention can be used in various administration forms such as oral and parenteral such as various injections, and can be realized in various dosage forms according to the administration form. For example, dosage forms include tablets, pills, capsules, granules, orally administered drugs such as suspensions, or solutions for intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, suspension injections, Freeze-dried preparations or suppositories,
Transmucosal dosage forms such as nasal and vaginal agents can be exemplified as typical ones.

As described above, in chemotherapy for infectious diseases and the like, in addition to drugs such as antibiotics and antibacterial agents, the defense function itself based on immune response such as resistance and activity of the patient is simultaneously improved. Therefore, it has been found that the addition and use of a component effective for this purpose is a very useful means clinically.

G-CSF, one of these components, is used in very small amounts. Therefore, when handling this as an extremely low-concentration aqueous solution or the like, it is often used, for example, put in a syringe or the like or housed in a container such as an ampoule. Because of its high adsorptivity to the walls of containers, syringes, etc., it is adsorbed to these walls, etc., to maintain the effective concentration in the drug solution or the desired activity of the components in the prescribed unit dose. Was difficult. Therefore, it is necessary to add an effective amount or more in advance in consideration of the amount lost by adsorption.

Furthermore, especially with regard to G-CSF, it is unstable, and is greatly affected by external factors such as temperature, humidity, oxygen, ultraviolet rays, etc., and physically and chemically by association, polymerization or oxidative decomposition. It causes a change and causes a decrease in activity.

Thus, the present invention has solved the above-mentioned problems by adding a protein to a G-CSF-containing preparation. Although the detailed mechanism of the stabilization and / or anti-adsorption effect of this is unknown, in the presence of protein, each G-CSF molecule is dispersed in the protein, and as a result, the interaction between G-CSF molecules is suppressed. The effect is highly mitigated, the probability of association, polymerization, etc. is greatly reduced, and at the same time, the adsorption site is reduced by competitively inhibiting the adsorption of G-CSF on the surface of the vessel by the protein. It is considered that the stability is significantly improved as an overall effect.

Such a problem is remarkable in injection solutions, suspensions, and the like, but is also a problem that is also observed in the process of preparing other tablets and the like. Is also effective.

That is, G-CSF is greatly stabilized by the addition of protein, and G-CSF is prolonged for a long time, as demonstrated in the Examples below.
The activity of F can be effectively maintained.

For these reasons, the amount of protein to be added is particularly critical at the lower limit, and 1 to 2 parts by weight per 1 part by weight of G-CSF.
It is desirable to contain it in an amount within the range of 0,000 parts by weight.

As described above, it is possible to effectively prevent adsorption to a vessel wall or the like and further improve stability, because G-
Since the CSF can be effectively used and waste of expensive components is prevented, the product cost can be reduced.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited at all by the following examples.

In the following examples, the measurement of the residual activity of G-CSF was carried out as follows.

(A) Soft agar method using mouse bone marrow cells 0.4 ml of horse serum, 0.1 ml of subject, 0.1 ml of bone marrow cell suspension of C3H / HeN (female) mouse (0.5 to 1 × 10 ⁵ nucleated cells),
0.4 ml of a modified McCoy's 5A culture solution containing 0.75% of agar was mixed and set in a plastic dish for tissue culture having a diameter of 35 mm, and then allowed to harden at 37 ° C, 5% carbon dioxide / 95% air, 100%
After culturing for 5 days under the condition of% humidity, the number of colonies formed (a colony composed of 50 or more cells is defined as one colony) is counted, and the activity of forming one colony is 1 unit (Unit)
And

The “modified McCoy” used in the above method (a) was used.
The “5A culture solution” was prepared as follows.

"Modified McCoy's 5A culture solution (2 times concentration)"McCoy's 5A culture solution (manufactured by GIBCO) 12 g, MEM
2.55 g of amino acid vitamin medium (manufactured by Nissui Pharmaceutical Co., Ltd.), 2.18 g of sodium bicarbonate, and 50,000 units of potassium penicillin G
After dissolving in 500 ml of distilled water, the solution was sterilized by filtration with a 0.22 μm Millipore filter before use.

(B) Reversed-phase high-performance liquid chromatography method Using a C8 reversed-phase column (4.6 mm × 300 mm, 5 μm), using n-propanol and trifluoroacetic acid as the mobile phase,
At least 1 μg equivalent of CSF is injected and the residual activity is measured under the following gradient conditions.

Solvent (A): 30% n-propanol, 0.1% trifluoroacetic acid Solvent (B): 60% n-propanol, 0.1% trifluoroacetic acid Measurement wavelength: 210 nm The residual amount of G-CSF measured by this method showed a very high correlation with the measurement result of the soft agar method using the mouse bone marrow cells described in (a) above.

Example 1 G-CS obtained by adding the proteins shown in Table 1 to 5 μg of G-CSF
A preparation containing F5 μg / ml (20 mM phosphate buffer, 100 mM sodium chloride, pH 7.4) was aseptically prepared, and then a lyophilized preparation was produced. The time course of the G-CSF activity was measured by the above-mentioned (a) soft agar method using mouse bone marrow cells. The results are shown in Table 1. The activity (%) in the table is a relative ratio to the initial unit, and is defined by the following formula.

The lyophilization conditions are as follows: The G-CSF solution to which the stabilizer has been added is placed in a sterile sulfur-treated glass vial and frozen at -40 ° C or lower for 4 hours.
Primary drying was performed at 40 ° C. to 0 ° C. and a degree of vacuum of 0.03 to 0.1 Torr for 48 hours. Next, 0 ° C to 20 ° C, vacuum degree 0.03 to 0.8 Torr
And the inside of the vial is replaced with sterile dry nitrogen gas until atmospheric pressure is reached. Then, it is stoppered with a rubber stopper for freeze-drying and sealed with an aluminum cap.

Example 2 GC obtained by adding the protein shown in Table 2 to 10 μg of G-CSF
A 10 μg / ml SF-containing preparation (20 mM phosphate buffer, 100 mM sodium chloride, pH 7.4) was aseptically prepared, aseptically filled in a sulfur-treated glass vial, sealed, and then subjected to G-CSF.
A solution formulation was prepared. For these solution preparations, G-
The time course of the CSF activity was measured in the same manner as in Example 1,
The results are shown in Table 2.

Example 3 GC obtained by adding the proteins shown in Table 3 to 10 μg of G-CSF
A preparation containing SF and 10 μg / ml (20 mM phosphate buffer, 100 mM sodium chloride, pH 7.4) was aseptically prepared, filled with 1 ml into a sulfa-treated silicone-coated glass vial, left at 4 ° C., The residual activity of G-CSF in the solution after 2 and 24 hours was measured by the reversed-phase high performance liquid chromatography method of the above (b) to determine the residual ratio (%).
The G-CSF adsorption preventing effect of the protein was evaluated. Table 3 shows the results.

Effects of the Invention As described above in detail, according to the present invention, by using a pharmaceutically acceptable protein in a predetermined amount, G-CSF present in a trace amount in the preparation, temperature, humidity, oxygen, It is possible to effectively solve problems relating to loss of active ingredient, decrease in activity, etc., which occur as a result of association, polymerization or oxidation or adsorption to container walls, etc. based on external factors such as ultraviolet rays. Was.

Therefore, it is possible to administer and control the dose of G-CSF to a patient extremely accurately, and furthermore, to use expensive G-CSF.
The CSF can be effectively used, and the cost of the G-CSF-containing preparation can be reduced.

 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-54-140707 (JP, A) JP-A-55-22604 (JP, A) JP-A-61-81781 (JP, A) JP-A-62 230729 (JP, A) JP-A-60-260523 (JP, A) JP-A-60-260522 (JP, A) JP-A-59-20225 (JP, A) JP-A-60-228422 (JP, A) JP-A-58-174330 (JP, A)

Claims (2)

(57) [Claims] 1. A group consisting of (1) human recombinant granulocyte colony-stimulating factor, and (2) human serum albumin, human serum globulin, gelatin, acid-treated or alkali-treated gelatin having an average molecular weight of 7,000 to 100,000, and collagen. At least one pharmaceutically acceptable protein selected from the group consisting of: a recombinant human granulocyte colony-stimulating factor-containing preparation; 2. The human recombinant granulocyte colony stimulating factor according to claim 1, wherein the protein is gelatin.