JPS6059000A - Method for stabilizing interferon - Google Patents

Abstract

PURPOSE:To improve remarkably the stability of interferon, having biological activities, e.g. antiviral and anticancer activities, and useful as medicines, etc. by bringing the interferon into contact with a polysaccharide. CONSTITUTION:A polysaccharide, e.g. dextran, chondroitin sulfuric acid, starch, glycogen, inulin, dextrin or an alginate, is added to stabilize any type, e.g. alpha,beta or gamma, of interferon such as natural interferon, interferon produced by the cultivation of animal cells, or produced by the cultivation of microorganisms obtained by recombination DNA techniques. The use of an inorganic salt, surfactant, blood serum albumin, antibiotic substance, chelating agent, amino acid and buffer agent, etc. together permits further improvement in stability.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing interferon and a composition containing interferon/and polysaccharide.

Interferon is a biologically active substance that is expected to be used as a medicine due to its biological activities such as antiviral and anti-motivational activities, and is divided into α-type, β-type, γ-type, etc. depending on its physiological and physicochemical properties and origin. are classified.

Conventionally, interferon has been produced by culturing animal, especially human, cells, but because supplies are limited, the interferon gene has been cloned and introduced into microorganisms, such as E. coli, using recombinant DNA technology. A method for producing interferon by culturing the microorganism has been developed.

Interferon is a protein or glycoprotein, but it is known to be an unstable substance.

Conventionally, in order to stabilize interferon, p11 linkage, addition of serum albumin, addition of soluble ranknide (Japanese Patent Application Laid-open No. 56-65822), addition of polyethylene nonionic surfactants, antibiotics, chelating agents, amino acids, etc. (Japanese Unexamined Patent Publication No. 58-92619), addition of acidic sugar (
JP-A No. 58-92621), addition of a sulfur-containing reducing agent (
JP-A-58-92622 1, Method of freeze-drying after adding H3A (human serum albumin) [Protein Nucleic Acid Enzyme Special Issue Advances in Interferon Research Vol. 25.
p, 358 f1981) Kyoritsu Shuppan], etc. are known.

New methods of stabilizing interferon are constantly being sought. As a result of various studies on methods for stabilizing interferon, it has been found that the stabilization of interferon is significantly increased by bringing interferon into contact with polysaccharides.

The present invention will be explained in detail below.

The interferons used in the present invention include natural interferons, interferons produced by culturing animal axes, interferons produced by culturing microorganisms obtained by recombinant DNA technology, and α.

Both β and T type interferons are included. The present invention particularly relates to interferon-β (hereinafter abbreviated as G-β-IFN) obtained by recombinant DNA technology.
Excellent effects can be obtained.

As polysaccharides, dextran [Dextran 40 (average molecular weight 40,000), Dextran 70 (average molecular weight 70,000)
] Chondroitin sulfate, starch.

Examples include glycogen, inulin, dextrin, alginate, etc.

In order to stabilize interferon, 5XIO-'' moles to 4x lQ-11 moles are added per unit of interferon (value determined based on international units and international standard samples, latest interferon - Asahi Science). Although it is effective if I
The effect is remarkable when it is added in proportion to -" moles to 2 X ] O-" moles.

Polysaccharides are effective when used alone, but their effects can be further enhanced by using them in combination with inorganic salts, surfactants, serum albumin, antibiotics, chelating agents, amino acids, buffers, and the like.

For example, when inorganic salts (sodium chloride, potassium chloride, magochloride, calcium chloride, etc.) are used together with polysaccharides, the amount of inorganic salts added is
The amount of the other additives is approximately the same as the amount of the inorganic salts, although the amount of the other additives varies depending on their molecular weight, etc., per unit.

Moreover, the effect of stabilizing interferon is further enhanced by freeze-drying the interferon-polysaccharide content rather than storing it in a solution state.

The invention also relates to compositions containing interferon and polysaccharides. In addition to the inorganic salts, the composition includes pharmaceutically acceptable preservatives, stabilizers, excipients, binders, disintegrants, wetting agents, lubricants, and coloring agents.

Flavoring agents, flavoring agents, coatings, suspending agents, emulsifying agents, solubilizing agents, buffering agents, isotonic agents, plasticizers, plastic surfactants, and the like can be included.

Examples of the present invention will be shown below.

Example 1 (i) Preparation of c-β-IFN G-β-IFN was prepared by the method described in JP-A-57-77654.
60% ethylene glycol-2M NaCl
An aqueous solution was prepared, and this was used as a G-β-IFN material.

(2) Sample preparation A polysaccharide, an inorganic salt, and a buffer were added to the above G-β-IFNIi material, and the mixture was freeze-dried. G-β-IFN 3XI
For O' units, dextrough 40 10.50.1
00 mg, NaC 16 g.

1/20M Na, HP O, - citrate buffer (
The equivalent of 2 ml of pH 4+ was used, and as a control, 50 ml of mannisotol was added instead of dextran 400.

(3) Measurement of interferon activity G-β-IFNfD titer +1, WISHmm* or FL
Inhibition of cell degeneration by vS virus using cells, dye uptake method (separate volume of protein and nucleic acid).

Progress in enzyme interferon research p355-363
(1981) and standard G-β-IFN [INTE
RFERON-β REFERENCE 5TANDA
RD (human recombinant Inoturf, onβ) Lot
K-Ref-04] was measured at the same time to determine the relative titer.

(4) Titer change due to freeze-drying (a) Additives shown in Table 1 and NaC16■.

1/20M Na, HP O, -phenoic acid tII solution (1) H4) Add 3XlO of G-β-IFN to 2+11 solution
' units were added, dissolved, and freeze-dried, and the residual ratio of biological activity after freeze-drying to that before freeze-drying was determined.

The results are not shown in Table 1.

Table 1 et al.) Changes in biological activity before and after freeze-drying were examined in the same manner as in (a) by changing the additives as shown in Table 2. The results are shown in Table 2.

Table 2 Example 2 Freeze-dried products were produced in the same manner as in Example 1 by varying the amount of sodium chloride, which is an inorganic salt, and their storage stability at 70°C was examined. The results are shown in Table 3.

Table 3 G-β-IFN 3X10@U Example 3 As shown in Table 4, the stability of the aqueous solution of the G-β-IFN raw material was investigated in a buffer solution and in distilled water. The results are shown in Table 4.

Table 4

Claims (2)

[Claims] (1) A method for stabilizing interferon, which comprises bringing interferon into contact with a polysaccharide. (2) A composition containing interferon and polysaccharide.