JPS61221129A - Stable gamma-interferon medicine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to stable lyophilized γ-interferon formulations that are clear and biologically active for at least 24 hours at room temperature upon reconstitution with water.

The term ``6γ-interferon''(GEF') refers to naturally occurring interferon or recombinant interferon that exhibits biological properties similar to those of human immune interferon. The word -A or GIF-A begins with cysteine-tyrosine-cystine and ends with glutamine.
Represents γ-interferon with 46 amino acids. Additionally, many incomplete interferon products are known, and the individual incomplete products are listed below.
γ-interferon is expressed in the same way by adding a capital letter after it. For example, GIF-D lacks the cysteine-tyrosine-cysteine fragment and thus has only 143 amino acids;
B is a model in which GIF-A is incomplete at the carboxyl terminus.
It is a fragment having 31 amino acids. Although all of these may be used in the present invention, the preferred form of GIF' for use in the present invention is GIFLA.

γ-interferon is a growth inhibitor, α-interferon and β-interferon (i.e. leukocyte interferon and fibroblast interferon)
It has been reported that it enhances the antiviral and antiproliferative effects of . It has also been suggested that γ-interferon can also serve as an immunoregulatory agent.

A known method for preparing interferon dosage forms consists of lyophilizing interferon in combination with other ingredients so that it can be prepared in water at the time of use. Unlike α- and β-interferon, which are stable at acidic pH, cy-interferon is known in the literature to be acid-labile, and thus has traditionally been treated at neutral or slightly alkaline pH. For example, UK patent application GB
2119313A has G per stirring of the prepared solution at pH 7.5.
Lyophilized formulations containing 7 to 23 micrograms of IF are disclosed. However, such neutral or slightly alkaline solutions at high concentrations of r-interferon (e.g. >0.2 mg/ml) cannot be used as injectable preparations as obvious precipitates immediately form. Such precipitates are undesirable for several reasons. For example, it may cause face-frame syndrome in the patient, reduce the efficacy of GIN', and may clog the injection needle.

Accordingly, the present invention provides a biologically stable lyophilized r-interferon formulation consisting of gamma-interferon and a compatible buffer, the buffer being a solution of 1 rr.
tl per 0.001mg preferably 0.05mg to 1
01! The solution is diluted with water to give a concentration of γ-interferon of up to 1.5 g, and is maintained at a pH in the range of 4.0 to 6.0, preferably 4.5 to 5.0.

Such formulations surprisingly constitute biologically and chemically stable lyophilized powders that form clear solutions when prepared in water.

Since GIF' is preferably administered parenterally, the formulations of the invention are primarily intended for injectable administration, ie intramuscular, intravenous and subcutaneous injection. However, if GIF solutions □ in other suitable dosage forms are required (e.g., topical dosage forms such as nasal sprays or eye drops), such formulations are also suitable for such other methods of administration of GIF. Will.

The acid instability of GIF is well documented. For example, J
, Journal by Vilcek et al.
of Immunological Methods,
69.61-70e1984 report the instability of GIF at low pH.

They recovered the main part of the column GIr eluted at pH 3.5, but as soon as this fraction was recovered, it was immediately placed in a neutral buffer medium.

At low pH, solutions of GIF' with or without acidic protein solubilizers such as human serum albumin are initially clear, but in neutral or slightly alkaline solutions the solutions become cloudy. Even at acidic pH, precipitation will eventually occur, but the presence of a solubilizing agent will significantly reduce the formation of precipitate.

The lyophilized powder of the present invention can be refrigerated at pH 4.5 for a long period of time (
For example, it is stable for one year). The prepared solution remains clear at room temperature or refrigerated for at least 24 hours, i.e. free of precipitates and/or
or does not produce turbidity and is biologically stable. Since the powder is prepared immediately before use, 24 hours is more than enough time to keep the solution clear for injection. Furthermore, the prepared solution remains clear and biologically stable after storage in the freezer for at least two weeks.

In the specification of the present invention, the term "biologically stable" means that the biological activity of the interferon preparation, as measured by standard methods using inhibition of the cytopathic effect (CPE) of the virus, is substantial on storage as described above. means that it is maintained. Other less quantitatively expressed biological functions, such as antiproliferative and immunoregulatory activities, all exhibit similar stability.

A significant advantage of the present invention is that GIF is more soluble at low pH, thus allowing higher concentrations than previously disclosed for neutral or alkaline formulations of GIF. . More than 0.2 mg G/ml at neutral pH
Although IF solutions produce precipitates, in the formulations of the present invention there can be up to 10 μg of GIF per 14 parts of clear prepared solution.

Another advantage of the present invention is that, due to the acidic pH of the formulation, it is possible to combine α- and/or β-interferon, both acid-stable acidic proteins, in the same formulation with r-interferon.

Yet another advantage is that the high concentrations of salts (eg, sodium chloride) that are often added as stabilizers to GIF solutions can be substantially reduced or eliminated. Lyophilization of low-salt GIF solutions produces lyophilized powders that are significantly superior to salt-containing solutions for preparing the dosage forms of the present invention. Such low salt formulations also form an isotonic solution suitable for intramuscular injection when prepared with water.

In the lyophilized preparation of the present invention, the buffer is present so that the pH of the prepared solution is 4.0 to 6.0, preferably 4.5 to 5.0. Preferred buffer systems are citric acid and disodium hydrogen phosphate (Na2HPO4), but other organic acid buffers such as succinic acid and tartaric acid and/or inorganic acids such as borax and one or more sodium phosphates may also be used. Buffers can also be used.

Preferably, the formulation includes a solubilizing agent, such as human serum albumin (H8A), gelatin or casein. H8A may be present in amounts up to 30 .eta./ml of prepared solution, but 1 .eta./yil is preferred.

When using antioxidants, examples include SH-specific reducing agents such as glutathione, thiodiglycolic acid, dithiothreitol and 2-mercaptoethanol, and ascorbic acid and cysteine or their pharmaceutically acceptable salts. A combination of can be used. A preferred antioxidant is a combination of ascorbic acid and cysteine hydrochloride-hydrate. GIF analogs (e.g. GI
In the case of FD), no antioxidant is required. Preferably, the antioxidant is present in the range of 0.1 to 5 mg/preparation solution.

Neutral amino acids (e.g. glycine, alanine), monosaccharides (e.g. glucose), disaccharides (e.g. lactose) and polyhydric sugar alcohols (e.g. one or more fillers such as mannitol) from 0 to 100/
ml preparation solution, preferably in the range of 10 to 30 ml/ml. A preferred filler is glycine.

Other excipients can also be added to the formulation at the option of those skilled in the art. For example, a chelating agent such as edetate disodium (disodium ethylenediaminetetraacetate) is added in a concentration range of 0 to 0.5 mg/ILtilJ eluent solution.

The above-mentioned solubilizer, antioxidant and filler are the G-factor of the present invention.
It is merely a preferred additive to the formulation. It is not necessary to use any or all of these alone or in combination to obtain the benefits of the present invention.

Carriers for the ready-to-use preparation of the lyophilized powder of the invention include, for example, water, isotonic water and solutions consisting of dextrose, glucose, propylene glycol and/or polyethylene glycol. If the prepared solution is not for injection (
For example, topical or oral solutions) use other pharmaceutically acceptable carriers such as ethanol. A suitable carrier for the preparation of the lyophilized powder of the invention is water.

Another aspect of the invention is the freezing of acidic bulk solutions of GIF'-
It is melt stable. The term "bulk solution" is 1-15
, preferably from 1 to 10 μGIF/ml and an antioxidant such as cysteine from 0 to 5 WIg/1 ILt, which aqueous solution is lyophilized G1. It was not prepared from I'. Up to 11! ! Freeze-thawing of a neutral bulk solution containing 9GIF'/ILJ produces a large amount of precipitate. However, to my surprise, the pH was 4.5-5.
5, preferably p) (In 5, even if the bulk solution contains 8 μg/ml of GIF, the temperature at the freezing temperature of the solution or lower (e.g. -10°C or lower, and the stability test at -120° C. It has been found that no precipitates form upon thawing even after long-term storage at a temperature of 0.9 °C (the customary temperature).

The buffer for the bulk solution is the same as the buffer described above used in the lyophilized formulation.

For example, such a bulk solution (pH 5) has the following composition:

G Engineering F' 2.0 Citric acid (anhydrous) 1.5 Disodium hydrogen phosphate (anhydrous) 1.8 Cystine hydrochloric acid hydrate 1.0 Sodium hydroxide (0,IN) Add as appropriate to adjust pH to 5 The use of frozen acidic Merck solution has several advantages for the storage of G-F'. For example, high concentrations of GIF at acidic pH are possible, which requires a small volume of storage solution and saves space. Furthermore, storing frozen products reduces the chance of contamination by microorganisms, for example, and is suitable for storing liquids. In addition, neutral 50% for transportation.
The conventional method of storing GIF' in % glycerol solution is not entirely satisfactory. This is because the glycerol solution freezes during transportation at the temperatures used for storage. The ability to freeze acidic bulk GIF' solutions simplifies subsequent handling, as it eliminates the need for costly and time-consuming dialysis to remove glycerol and concentrate the solution before use. Similarly, the use of acidic bulk solutions eliminates the high concentrations of salts, such as sodium chloride, traditionally used to stabilize GIF' solutions, further simplifying pre-use procedures in formulations. do.

The following example describes a method for preparing a sterile powder for use in preparing an injectable solution of r-interferon, but the invention is not limited thereto.

Example (q/) Koial) (mr/)9al) G I
F' *0.5 1.0 Aminoacetic acid (glycine) 20 20 Citric acid (anhydrous) 1.5 1.5 Hydrogen disodium (anhydrous) 1.8
1.8 Edetate disodium 0.
1 0.1 ascorbic acid
22cysti Δbasic acid salt hydrate 0.75
0.5 Human Serum Albumin 11 Water for Injection 1ml 1ml 1m
l Combine the above ingredients and lyophilize using standard techniques.

*G-F is provided as a bulk solution as described above, particularly as exemplified above. The final concentration of cysteine hydrochloride is therefore 1.0 μ/vial in each formulation.

Claims (24)

[Claims] (1) A biologically stable lyophilized γ-interferon preparation consisting of γ-interferon and a compatible buffer, the buffer being 0.001 mg/ml of solution;
Preferably, the above formulation maintains the pH of the solution prepared in water in the range of 4.0 to 6.0 to give a concentration of γ-interferon of from 0.05 mg to 10 mg. (2) The formulation according to claim 1, further comprising a solubilizer. (3) A formulation according to claim 2, wherein the solubilizing agent is up to 30 mg of human serum albumin per ml of prepared solution. (4) The formulation further comprises an SH-specific reducing agent and an antioxidant selected from a combination of ascorbic acid and cysteine or a pharmaceutically acceptable salt thereof. The formulation according to any one of the above. (5) The formulation according to claim 4, wherein the antioxidant is a combination of ascorbic acid and cysteine. (6) According to any one of claims 1 to 5, the preparation contains 0 to 100 mg of one or more fillers selected from neutral amino acids, monosaccharides, disaccharides, and polyhydric sugar alcohols. The formulation described. (7) The formulation according to claim 6, wherein the filler is glycine. (8) The formulation according to any one of claims 1 to 7, further comprising a chelating agent. (9) Claim 8 in which the chelating agent is disodium edetate (disodium ethylenediaminetetraacetate)
Preparations described in section. (10) The formulation according to any one of claims 1 to 9, wherein the buffer contains citric acid and disodium hydrogen phosphate. (11) The formulation according to claim 10, wherein the buffer maintains a pH of 4.5 to 5.0 in the prepared solution. (12) γ-interferon is 0.1-2 mg/ml
12. A formulation according to any one of claims 1 to 11, which is present in a prepared solution. (13) The preparation according to claim 12, wherein the γ-interferon is γ-interferon-A. (14) The formulation according to any one of claims 1 to 13, further comprising α- or β-interferon. (15) 0.05-10 mg γ-interferon/m
l preparation solution, buffer system consisting of citric acid and disodium hydrogen phosphate to maintain a pH range of 4.0-6.0, 0-30 mg human serum albumin/ml preparation solution, 0
．． 1-5 mg ascorbic acid and 0.1-5 mg cysteine/ml preparation solution, 0-100 mg glycine/ml
Preparation solution and 0-0.5 mg edetate disodium/
Formulation with ml preparation solution. (16) 0.1-2 mg γ-interferon/ml prepared solution, buffer system consisting of citric acid and disodium hydrogen phosphate to maintain pH at 4.5, 1 mg human serum albumin/ml prepared solution, 2 mg ascorbine 16. A formulation according to claim 15 having an acid and 1 mg cysteine/ml prepared solution, 20 mg glycine/ml prepared solution and 0.1 mg edetate disodium/ml prepared solution. (17) The formulation according to any one of claims 1 to 16, which has a prepared solution of 1 mg γ-interferon/ml. (18) The preparation according to any one of claims 1 to 17, wherein the γ-interferon is γ-interferon-A. (19) The formulation according to any one of claims 1 to 18, which is suitable for injection. (20) 1-10 mrγ-interferon and 0-
5 mg antioxidant/ml solution, and the pH of the solution to pH 4.
．． A method for storing GIF comprising storing a solution containing a compatible buffer maintaining a temperature in the range of 5 to 5.5, preferably about 5.0, at or below its freezing temperature. (21) The method according to claim 20, wherein the buffer contains citric acid and disodium hydrogen phosphate. (22) Claim 2 in which the pH of the solution is 5.0
0 or 21. (23) The method according to any one of claims 20 to 22, wherein the antioxidant is cysteine. (24) The solution according to any one of claims 20 to 23, which is stored at freezing temperature or lower.