Method for solubilization of interferon

A method for increasing the solubility of interferon in water involves admixing an amino acid selected from the group consisting of arginine, histidine, lysine, hydroxylysine, ornithine, glutamine, .gamma.-aminobutyric acid, .epsilon.-aminocaproic acid and a salt thereof with interferon.

BACKGROUND OF THE INVENTION 
The present invention relates to a method for effecting solubilization of 
interferon in water and an interferon-containing composition which is 
easily soluble in water. 
Interferon is a physiologically active substance expected to be useful as a 
therapeutic agent because of its biological activities such as antivirus, 
anti-cancer and like activities. Interferon is classified into type 
.alpha., type .beta., type .gamma., etc., according to the physiological 
and physicochemical properties, and the origin. Heretofore, interferon has 
been produced by incubating cells of animals, in particular, cells of 
humans. However, 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., E. coli, and incubating the microorganism. 
Interferon is only slightly soluble in water so that it is difficult to 
isolate and purify from the culture. Thus, a procedure for solubilizing 
interferon has been sought. 
For the purification of interferon-.beta., a method using blue Sepharose 
column is known. In this method, interferon is solubilized using ethylene 
glycol in an eluate [Jankowski, W. J., et al, Biochemistry, 15, 5182 
(1976), Knight, E., Jr., Science, 207, 525 (1980)], but adequate results 
from this method have not been obtained yet. 
Furthermore, Japanese Published Unexamined Patent Application No. 
102519/1980 discloses that aromatic amino acids such as tryptophan, 
phenylalanine, tyrosine, etc., are effective for stabilizing interferon. 
However, aromatic amino acids are not readily soluble in water and, 
therefore, are undesirable as reagents for solubilizing interferon. 
SUMMARY OF THE INVENTION 
The present invention provides a method for increasing the solubility of 
interferon in water using an amino acid selected from the group consisting 
of arginine, histidine, lysine, hydroxylysine, ornithine, glutamine, 
.gamma.-aminobutyric acid, .epsilon.-aminocaproic acid, and a salt thereof 
such as hydrochloride, sulfate, acetate, glutamate, aspartate and maleate. 
DESCRIPTION OF THE INVENTION 
Examples of interferon include natural interferon, interferon produced by 
incubation of animal cells, interferon produced by incubation of a 
microorganism obtained by recombinant DNA technology, etc. Also included 
are interferons of any of types .alpha., .beta. and .gamma.. In the 
present invention, in particular, an excellent effect may be expected with 
interferon-.gamma. (hereinafter referred to as G-.gamma.-IFN) obtained by 
recombinant DNA technology. 
As the amino acids, arginine, histidine, lysine, hydroxylysine, ornithine, 
glutamine, .gamma.-aminobutyric acid, .epsilon.-aminocaproic acid, a salt 
thereof, etc., can be used. In particular, arginine, histidine, lysine, 
hydroxylysine and ornithine have been found to provide a remarkable 
solubilizing effect. 
The effect of solubilizing interferon is exhibited by adding 
5.times.10.sup.-6 mole to 5.times.10.sup.-3 mole of the amino acid per 
1,000,000 units of interferon. The effect is especially remarkable with 
the addition of the amino acid in an amount of 2.5.times.10.sup.-5 mole to 
2.times.10.sup.-3 mole. 
When the amino acids are used in combination with serum albumin; inorganic 
salts such as sodium chloride, potassium chloride, sodium carbonate, 
sodium bicarbonate, potassium phosphate, potassium phosphate monobasic, 
potassium phosphate dibasic, potassium phosphate tribasic, sodium 
phosphate, sodium phosphate monobasic, sodium phosphate dibasic, sodium 
phosphate tribasic, sodium tetraborate and potassium tetraborate; 
polysaccharides such as chondroitin sulfate, sodium 
carboxymethylcellulose, dextran, dextrin, cyclodextrin, methylcellulose 
and ethylhydroxycellulose; surfactants such as benzalkonium chloride, 
benzethonium chloride, sodium lauryl sulfate, Tween 80, Tween 60, Span 60, 
Span 40 and Span 20; chelating agents such as edetate disodium, citric 
acid and glycine, etc., the effect can be markedly increased. Generally 
the amount of such other additive will vary from 1.times.10.sup.-8 mole to 
1.times.10.sup.-2 mole per 1,000,000 units of interferon depending on the 
specific additive selected. 
The conventional, freeze-dried, serum albumin-added G-.gamma.-IFN gives 
white insoluble matters in a solution at 25.degree. C. six hours after it 
is dissolved in water. However, when G-.gamma.-IFN is dissolved using a 3% 
(W/V) amino acid solution, insoluble matter is not formed at 25.degree. C. 
even after six hours. 
Further, when freeze-dried G-.gamma.-IFN to which an albumin and an amino 
acid are added is dissolved in water, insoluble matter is not formed at 
25.degree. C. even after six hours. 
The present invention also provides an interferon-containing composition 
containing an amino acid as a solubilizing agent. 
The amounts of interferon, the amino acids and the inorganic salts in the 
composition are the same as in the aforesaid method for solubilization. 
Such compositions may incorporate pharmaceutically acceptable 
preservatives, stabilizers, excipients, binding agents, 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.

Examples of the present invention are given hereinafter: 
EXAMPLE 1 
Preparation of a sample: 
In this example, 5 mg of serum albumin and 3.times.10.sup.6 units of 
G-.gamma.-IFN which was prepared by the method described in Reference 
Example below (hereinafter the same method of preparation shall apply) 
were dissolved in 2 ml of distilled water and the solution was 
freeze-dried. 
Measurement of Insoluble Matter: 
The amount of the insoluble matters in a reconstructed solution of the 
freeze-dried G-.gamma.-IFN was determined by measuring the absorbance at 
400 nm. 
The reconstruction was carried out using 5 ml of the amino acid solution 
shown in Table 1 and an equal amount of distilled water was used as a 
control. After reconstruction, the solution was stored for six hours at 
25.degree. C. and then the solution was put in a 1 cm quartz cell, where 
the absorbance was measured at 400 nm. 
The results of different solutions are shown in Table 1. 
TABLE 1 
______________________________________ 
Amino acid Concentra- O.D. 400 nm at 25.degree. C., 
solution tion (W/V %) 6 hours after 
______________________________________ 
Distilled water 0.260 
(control) 
Arginine 3 0.026 
monohydrochloride 
Lysine 3 0.045 
monohydrochloride 
Histidine 3 0.034 
Hydroxylysine 
3 0.043 
Glutamine 3 0.063 
Ornithine acetate 
3 0.042 
.gamma.-Aminobutyric acid 
3 0.077 
.epsilon.-Aminocaproic acid 
3 0.071 
Propylene glycol 
5 0.231 
______________________________________ 
EXAMPLE 2 
In this example, 5 mg of serum albumin, 50 mg of an amino acid shown in 
Table 2 and 3.times.10.sup.6 units of G-.gamma.-IFN were dissolved in 2 ml 
of distilled water, and the solution was freeze-dried. 
The freeze-dried product was dissolved in 5 ml of distilled water. The 
solution was stored for six hours at 25.degree. C., and then the 
absorbance of the solution was measured at 400 nm. 
The results of different amino acids are shown in Table 2. 
TABLE 2 
______________________________________ 
Amino Acid O.D. 400 nm 
______________________________________ 
None 0.255 
Arginine monohydrochloride 
0.029 
Lysine monohydrochloride 
0.042 
Histidine 0.028 
Ornithine acetate 0.034 
______________________________________ 
EXAMPLE 3 
The freeze-dried G-.gamma.-IFN was dissolved in a manner similar to Example 
1 except that the concentrations of the amino acids in the solution were 
changed. The solution was stored for six hours at 25.degree. C., and then 
the absorbance of the solution was measured at 400 nm. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Concentra- 
Amino Acid tion (W/V %) 
O.D. 400 nm 
______________________________________ 
Distilled water 0.259 
(control) 
Arginine 4 0.026 
monohydrochloride 
Arginine 1 0.047 
monohydrochloride 
Arginine 0.5 0.093 
monohydrochloride 
Lysine 1 0.061 
monohydrochloride 
Histidine 1 0.045 
Orinithine acetate 
1 0.080 
______________________________________ 
EXAMPLE 4 
In this example, 5 mg of serum albumin and 3.times.10.sup.6 units of 
G-.gamma.-IFN were dissolved in 2 ml of distilled water, and the solution 
was freeze-dried. The freeze-dried product was dissolved in 5 ml each of 
distilled water, a solution of albumin, and a solution mixture of albumin 
and an amino acid. The solution was stored for six hours at 25.degree. C., 
and then the absorbance of the solution was measured at 400 nm. The 
results are shown in Table 4. 
TABLE 4 
______________________________________ 
Solution O.D. 400 nm 
______________________________________ 
Distilled water 0.256 
Solution of serum albumin (4 mg/ml) 
0.208 
Serum albumin (4 mg/ml), arginine 
0.025 
monohydrochloride (3 w/v %) 
______________________________________ 
EXAMPLE 5 
In this example, 5 mg of serum albumin, 5 mg of sodium chloride and 30 mg 
of arginine monohydrochloride were added to 3.times.10.sup.6 units of 
G-.gamma.-IFN. The solution was made up to 2 ml with distilled water and 
put in a vial, followed by freeze-drying. 
The freeze-dried product was dissolved in 5 ml of distilled water. The 
solution was stored for six hours at 25.degree. C., and then the 
absorbance of the solution was measured at 400 nm. The O.D. value was 
0.025. The amount of the G-.gamma.-IFN which remained in the solution 
after six hours was 98% of the expected value (3.times.10.sup.6 units). 
REFERENCE EXAMPLE 
Production of interferon-.gamma. with Escherichia coli IGKA-2: 
Escherichia coli IGKA-2 (FERM BP-496) having recombinant plasmid pGKA-2 was 
cultivated at 37.degree. C. for 18 hours in LG medium prepared by 
dissolving 10 g of tryptophan, 5 g of yeast extract, 5 g of NaCl and 2 g 
of glucose in 1 liter of water and adjusting the pH to 7.0 with NaOH. The 
culture (4 ml) was inoculated on 200 ml of MCG medium (0.6% Na.sub.2 
HPO.sub.4, 0.3% KH.sub.2 PO.sub.4, 0.5% NaCl, 0.1% NH.sub.4 Cl, 0.5% 
glucose, 0.5% casamino acid, 1 mM MgSO.sub.4, and 4 .mu.g/ml vitamin 
B.sub.1, pH 7.2). After incubating at 30.degree. C. for 4 to 8 hours, 10 
.mu.g/ml indolylacrylic acid, which was an inducer of a tryptophan gene, 
was added to the culture. Incubation was continued for further 2 to 12 
hours. The culture was centrifuged at 8,000 rpm for 10 minutes to collect 
cells, and the cells were washed with 30 mM NaCl and 30 mM Tris-HCl (pH 
7.5) buffer. The washed cells were suspended in 20 ml of the aforesaid 
buffer, and 4 mg of lysozyme and 0.1 ml of 0.25 M EDTA 
(ethylenediamine-tetraacetic acid) were added to the suspension. After the 
mixture was allowed to stand at 0.degree. C. for 30 minutes, freezing and 
thawing were repeated 3 times to disrupt the cells. The mixture was 
centrifuged at 15,000 rpm for 30 minutes to obtain the supernatant. The 
supernatant was treated through precipitation with ammonium sulfate, gel 
filtration with Sephadex G-75, ion exchange chromatography, etc., to 
obtain about 2.times.10.sup.6 units of interferon-.gamma..