Method for stabilizing interferon.

0001The present invention relates to a method for stabilizing interferon and to compositions which contain interferon and a saccharide or inorganic salt for stabilizing the interferon.

0002Interferon is a physiologically active substance useful as a therapeutic agent because of its biological activities such as antivirus, anti-cancer and like activities. Interferon is classified into type a, type S, type y, etc., according to the physiological and physicochemical properties, and the origin.

0003Interferon has been produced by incubating cells of animals, in particular, cells of humans, but since the supply of such cells was limited, there has been developed a process for producing interferon which comprises cloning an interferon gene by recombinant DNA technology, introducing it into a microorganism; e.g., Escherichia coli, and incubating the microorganism.

0004Interferon is a protein or a sugar protein which is unstable and its biological activity is decreased during clinical application. Heretofore, it is known that pH adjustment, addition of serum albumin, addition of soluble lanthanide or calcium salts (U.S. Patent 4,252,791), addition of polyethylenic nonionic surfactants, antibiotics, carboxylic acid, chelating agents, amino acids, etc. (Japanese Published Unexamined Patent Application No. 102519/1980), addition of polyethylenic nonionic surfactants, antibiotics, chelating agents, amino acids, etc., addition of acid sugars and addition of sulfur-containing reducing agents (European Patent Publication No. 80879), addition of HSA (human serum albumin) and, subsequently, freeze-drying it (Progress of Interferon Research, extra issue for "Protein, Nucleic Acid and Enzyme," No . 25, p. 358 (1981), Kyoritsu Shuppan), and so forth, are effective for stabilizing interferon. However, there is always a demand for a new method for stabilizing interferon. As the result of intensive studies of a method for stabilizing interferon, it has been found that the stability of interferon is remarkably enhanced by admixing the interferon with an inorganic salt such as sodium chloride, potassium chloride, etc., either alone or in combination with, for example, serum albumin.

0005Further, it has been found that the stability of interferon can also be remarkably increased by bringing interferon into contact with a saccharide alone or admixed with other additives.

0006Examples of interferon used in the present invention include natural interferon, interferon produced by incubation of animal cells, interferon produced by incubation of a microorganism obtained by recombinant DNA technology, etc., and also interferon of any of types a, a and y may be included. In the present invention, in particular, an excellent effect may be expected with Interferon-a (hereinafter referred to as G-α-IFN) obtained by recombinant DNA technology.

0007As the non-toxic inorganic salt, alkali metal salt, such as sodium chloride, potassium chloride and lithium chloride, or magnesium chloride is used, and in particular, sodium chloride and potassium chloride are remarkable in the stabilizing effect.

0008In order to stabilize interferon, the inorganic -salt is formulated; i.e., admixed with the interferon, in an amount of 1x10^-11 - 3 x 10^-10 mole per unit of interferon (international unit; "Latest Interferon" compiled by Kotaro Kishida, Asahi Science 1981), preferably, 3 x 10^-11 - 5 x 10^-11 mole per unit of interferon.

0009The inorganic salt exhibits a remarkable effect even when used alone and the effect may be even further enhanced by using it in-combination with, for example, serum albumin, surfactants, antibiotics, chelating agents, amino acids, etc.

0010The conventional G-β-IFN freeze dried preparation employing serum albumin as a stabilizer becomes insoluble in water in 7 days or 14 days at 70°C, and its biological activity is reduced to 50-7%; whereas a G-6-IFN preparation containing serum albumin and sodium chloride can be readily dissolved in water in the same period of time, and about 90% of its biological activity remains.

0011Furthermore, the stability of G-B_-IFN in an aqueous solution is examined by measuring the biological activity of 3 x 10⁶ units/ml G-B-IFN under a temperature condition of 40°C. In the case where serum albumin is used, the G-B-IFN activity is reduced to 20% after 6 hours. On the other hand, where serum albumin and sodium chloride are used, 40% the G-6-IFN activity still remains after 6 hours.

0012As mentioned above, the stabilizing effect is achieved by bringing interferon. into contact with a saccharide. Examples of a useful saccharide include dextran; e.g., dextran 40 (average molecular weight 40,000), and dextran 70 (average molecular weight 70,000), chondroitin sulfuric acid, starch, glycogen, inulin, dextrin, alginic acid salts, etc.

0013In order to stabilize interferon, the saccharide is formulated with the interferon in an amount of 5 x 10^-14 - 4 x 10^-12 mole per unit of interferon (international unit: a value determined based on an international standard sample; "Latest Interferon" in Asahi Science 1981), pre- _fer_ably, 1 x 10^-13 - 2 x 10^-12 mole per unit of interferon.

0014The saccharide exhibits an effect even when used alone, and the effect may be even further enhanced by using it in combination with inorganic salts, surfactants, serum albumin, antibiotics, chelating agents, amino acids, buffers, and the like additives which are non-toxic and pharmaceutically acceptable additives.

0015For example, when an inorganic salt (e.g., sodium chloride, potassium chloride, magnesium chloride, calcium chloride, etc.) is to be used in combination with the saccharide, the amount of the inorganic salt added is 1 x 10^-11 - 3 x 10^-10 mole per unit of interferon, and the amounts of the other additives may be, more or less, the same as that of the inorganic salt, although the amount to be added is dependent on their molecular weights, etc.

0016Further, the effect of a saccharide to stabilize interferon is more enhanced by storing a preparation containing interferon and a saccharide in a freeze-dried state rather than in a solution state.

0017The present invention also relates to compositions which contain interferon, and an inorganic salt or a saccharide. The amount of an inorganic salt or a saccharide to be contained in the compositions is the same as described for the above stabilizing method. Such compositions may incorporate, in addition to the inorganic salts or saccharides, pharmaceutically acceptable preservatives, stabilizers, binding agents, excipients, disintegrating agents, wetting agents, lubricants, coloring agents, aromatic agents, flavoring agents, coating agents, suspending agents, emulsifiers, dissolution aids, buffers, isotonic agents, plasticizers, plastic surfactants, etc.

0018Examples of the present invention are given below.

Example 1

(1) Preparation of G-β-IFN

0019Purified G-β-IFN was prepared according to the procedures described in European Patent Publication No. 42246, published on December 23, 1981 and about 200 µg of serum albumin (HSA: produced by CRYOSAN or BSA: produced by Seikagaku Kogyo) was added to about 5 µg of purified G-6-IFN.

(2) Preparation of Samples

0020Serum albumin and, as a stabilizer, an inorganic salt such as sodium chloride were added to the aforesaid G-β-IFN, and the mixture was freeze-dried. The preparation of G-β-IFN containing 10 mg of serum albumin and 5 mg of sodium chloride was used based on 3x10 units of G-β-IFN, while, as a control, that obtained by adding serum albumin alone was used.

0021One ml of distilled water were added to each of the freeze-dried products to prepare samples of aqueous solution.

(3) Measurement of Interferon Activity

0022The potency of G-B-IFN was measured to determine the relative potency by simultaneously measuring standard G-β-IFN using a cytophatogenic inhibition - dye inclusion method by VSV virus employing WISH cells or FL cells (Progress of Interferon Research, extra issue for "Protein, Nucleic Acid and Enzyme," No. 25, p. 355-363 (1981), Kyoritsu Shuppan).

(4) Stability Tests

(a) Freeze-dried preparations

0023A storage stability test was conducted with the preparations having the compositions set forth in Table 1 in 7 days and 14 days in a constant temperature cell at 70°C.

0024The results are shown in Table 1. <tables id="tabl0001" num="0001"><img file="EP0123291A2_D0001.tif" /></tables>

(b) Aqueous solutions

0025The freeze-dried preparations having the compositions set forth in Table 1 were dissolved in 1 ml of distilled water, put in a constant temperature cell at 40°C, and the potency in 6 hours and 24 hours were measured.

0026The results are shown in Table 2. <tables id="tabl0002" num="0002"><img file="EP0123291A2_D0002.tif" /></tables>

Example 2

0027The storage stability of the G-β-IFN freeze-dried preparations was examined in the same manner as in Example 1 except that the amounts of the inorganic salts were changed to those given in Table 3.

0028The results are shown in Table 3. <tables id="tabl0003" num="0003"><img file="EP0123291A2_D0003.tif" /></tables>

Example 3

0029The storage stability of the freeze-dried preparations at 70°C was measured in the same manner as in Example 1 by using 6 x 10⁶ units of purified G-β-IFN containing D-mannitol and inorganic salts as stabilizers.

0030The results are shown in Table 4. <tables id="tabl0004" num="0004"><img file="EP0123291A2_D0004.tif" /></tables>

Example 4

0031The storage stability in an aqueous solution at 40°C was measured by using 1.8 x 10⁷ units of purified G-β-IFN containing 30 mg of NaCl or KC1 as a stabilizer. The results are shown in Table 5. <tables id="tabl0005" num="0005"><img file="EP0123291A2_D0005.tif" /></tables>

Example 5

(1) Preparation of G-β-IFN

0032G-β-IFN was prepared according to the procedures described in European Patent Publication No. 42246, published on December 23, 1981 and made into a 60% ethylene glycol - 2M NaCl aqueous solution, which was used as a G-β-IFN (2 x 10⁸ U/ml).

(2) Preparation of Samples

0033A saccharide, an inorganic salt and a buffer were added to the aforesaid G-B-IFN solution and the mixture was freeze-dried. The preparation of G-B-IFN containing each of 10, 50 and 100 mg of dextran 40, 6 mg of NaCl and 2 ml of a 1/20 M Na₂HPO₄ - citric acid buffer (pH 4) based on 3 x 10⁶ units of G-β-IFN was used while, as a control, a preparation containing the NaCl, the buffer and 50 mg of mannitol instead of dextran 40 was used.

(3) Measurement of Interferon Activity

0034The potency of G-6-IFN was measured according to the same procedure as Example 1 (3).

(4) Change in Potency by Freeze-Drying

0035(a) 3 x 10⁶ units of the G-β-IFN was added to each of solutions containing the respective additives set forth in Table 6, 6 mg of NaCl and 2 ml of a 1/20 M Na₂HPO₄ - citric acid buffer (pH 4), and the mixture was subjected to freeze-drying. Freeze-dried samples were dissolved in water and diluted in Eagle's minimal essential medium supplemented with 5% fetal bovine serum. The percent of the biological activity remaining after the freeze-drying relative to the biological activity before the freeze-drying was determined. The results are shown in Table 6. <tables id="tabl0006" num="0006"><img file="EP0123291A2_D0006.tif" /></tables>

0036(b) Upon changing the additives, as set forth in Table 7, the change in biological activity before and after the freeze-drying was again determined.

0037The results are shown in Table 7. <tables id="tabl0007" num="0007"><img file="EP0123291A2_D0007.tif" /></tables>

Example 6

0038Freeze-dried samples were produced in the same manner as in Example 5 after changing the amount of sodium chloride used as the inorganic salt, and the storage stability at 70°C was studied.

0039The results are shown in Table 8. <tables id="tabl0008" num="0008"><img file="EP0123291A2_D0008.tif" /></tables>

Example 7

0040The stability of an aqueous solution of 1.5x106 U/ml G-β-IFN prepared in Example 5 (1) was studied both in a buffer and in distilled water.

0041The results are shown in Table 9. <tables id="tabl0009" num="0009"><img file="EP0123291A2_D0009.tif" /></tables>