JP2799483B2 - Method for stabilizing interleukin-1β composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to interleukin-1β.
The present invention relates to a method for stabilizing a composition containing an (IL-1β) active substance.

[0002]

2. Description of the Related Art It is known that IL-1β is produced not only from macrophages and monocytes but also from many cells and exhibits various biological activities [Metabolism, Vol. , p97-104 (1986); Medical Immunology, Vol. 12, N
o.6, p753-760 (1986) etc.].

[0003] Based on the above activity, the IL-1β is expected to be applied as a pharmaceutical.
β and its derivatives have been shown to be effective for various pharmaceutical uses [European Patent Publication No. EP0237
967A2].

[0004] On the other hand, when applied as a pharmaceutical, the active ingredient is converted under ordinary pharmaceutical forms and storage conditions.
Needless to say, it is required to be stable without changing over time, and the above-described performance is naturally required also in the case of IL-1β. In particular, in the case of a highly purified homogenous product which is highly purified before clinical application is possible, sufficient consideration is required for its handling in terms of stability, and its activity is usually restricted in various ways. Therefore, there is a need in the art for the development and improvement of a stabilized pharmaceutical composition of IL-1β which can be more stably maintained under various storage conditions such as freezing treatment, freeze-drying treatment, temperature, and time. I have.

[0005]

The IL-1β active substance has a strong pharmacological activity and is used in an extremely small amount. On the other hand, it has an adsorptive property to the container wall, and the lower the content as an active ingredient, the lower the activity. This adsorptivity becomes a problem, and its prevention is required. Further, IL-1β activity is unstable, and this instability tends to increase as the content of the active ingredient decreases.

The present invention solves the above problems and provides a stable composition of an IL-1β active substance which is particularly suitable for application as a medicament, and further provides an isotonic IL-1β active substance. It is an object of the present invention to provide a stabilized composition.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies on the above-mentioned objects, and as a result, have found that a composition containing an IL-1β active substance is obtained.
When human serum albumin is blended with (lyophilized form), it exhibits an adsorption preventing effect and a stabilizing effect.
Found that the activity of 1β-active substance was remarkably stabilized,
Here, the present invention has been completed.

According to the present invention, there is provided a method for stabilizing a lyophilized form of an IL-1β composition, which comprises human serum albumin together with an IL-1β active substance.

In the present invention, IL-1β active substances used as active ingredients include IL-1β and its derivatives. More specifically, such active ingredients include European Patent Publication (EP023796) of the applicant of the present invention.
7A2) and equivalents thereof. The polypeptide has the following formula (A): Ala-Pro-Val-Arg-Ser-Leu-Asn-Cys-Thr-Leu-Arg-Asp-Ser-Gln-Gln-Lys-Ser-Leu-Val-Met- Ser-Gly-Pro-Tyr-Glu-Leu-Lys-Ala-Leu-His-Leu-Gln-Gly-Gln-Asp-Met- Glu-Gln-Gln-Val-Val-Phe-Ser-Met-Ser- Phe-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-Ile-Pro-Val-Ala-Leu-Gly-Leu-Lys-Glu-Lys-Asn-Leu-Tyr-Leu-Ser- Cys-Val- Leu-Lys-Asp-Asp-Lys-Pro-Thr-Leu-Gln-Leu-Glu-Ser-Val-Asp-Pro-Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met- Glu-Lys-Arg-Phe-Val-Phe-Asn-Lys-Ile-Glu-Ile-Asn-Asn-Lys-Leu-Glu-Phe-Glu-Ser-Ala-Gln-Phe-Pro-Asn-Trp- Tyr-Ile-Ser-Thr-Ser-Gln- Ala-Glu-Asn-Met-Pro-Val-Phe-Leu-Gly-Gly-Thr-Lys-Gly-Gly-Gln-Asp-Ile-Thr-Asp- In the amino acid sequence of IL-1β represented by Phe-Thr-Met-Gln-Phe-Val-Ser-Ser, a) 1st position Ala, 3rd position Val, 4th position Arg, 5th position Ser, 8th position Cy
s, Arg at position 11, His at position 30, Cys at position 71, Ly at position 93
s, Lys at position 97, Arg at position 98, Phe at position 99, Ly at position 103
s, at least one amino acid residue selected from Trp at position 120, Tyr at position 121 and Ser at position 153 has been deleted or replaced with another amino acid residue; b) 9 to 9 amino acids from Ala at position 1 The amino acid sequence leading to the position Thr or at least one amino acid residue in the amino acid sequence is deleted (provided that the above-mentioned a) has Ala, 3-Val, 4-A
rg, unless at least one of the amino acid residues selected from the group consisting of Ser at position 5, Ser at position 8 is deleted), c) the amino acid sequence from Lys at position 103 to Ser at position 153, or At least one amino acid residue is deleted (provided that the Lys at position 103, 12
Excluding the case where at least one amino acid residue selected from the group consisting of Trp at position 0, Tyr at position 121 and Ser at position 153 is deleted), and d) an amino acid residue at the N-terminus of the above formula (A) Or the following formula (B)
(B) Met-Ala-Glu-Val-Pro-Glu-Leu, wherein the amino acid sequence from Met at position 1 'to Asp at position 116 or a partial amino acid sequence at the C-terminal thereof is added. -Ala-Ser-Glu-Met-Met-Ala-Tyr-Tyr-Ser-Gly-Asn-Glu-Asp-Asp-Leu-Phe-Phe-Glu-Ala-Asp-Gly-Pro-Lys-Gln-Met -Lys-Cys-Ser-Phe- Gln-Asp-Leu-Asp-Leu-Cys-Pro-Leu-Asp-Gly-Gly-Ile-Gln-Leu-Arg-Ile-Ser-Asp-His-His-Tyr -Ser-Lys-Gly-Phe-Arg-Gln-Ala-Ala-Ser-Val-Val-Val-Ala-Met-Asp-Lys-Leu-Arg-Lys-Met-Leu-Val-Pro-Cys-Pro -Gln-Thr-Phe-Gln-Glu-Asn-Asp-Leu- Ser-Thr-Phe-Phe-Pro-Phe-Ile-Phe-Glu-Glu-Glu-Pro-Ile-Phe-Phe-Asp-Thr -Trp-Asp-Asn-Glu-Ala-Tyr-Val-His-Asp characterized by having an amino acid sequence that satisfies at least one of the conditions a) to d).

The designations of amino acids and polypeptides hereinbefore and hereinafter refer to IUPAC and IUAC-
Abbreviations in IUB nomenclature or rules or abbreviations commonly used in the art shall be followed.
The same applies to the display of the nucleic acid in the base sequence. The number or position of amino acids is indicated in accordance with the amino acid sequence of IL-1β, that is, the sequence of the above formula (A), even if there are deletions and additions. However, those with a dash in the numerical value indicating the position of the amino acid follow the amino acid sequence of the formula (B).

Hereinafter, the polypeptide (IL-1β and its derivatives) will be described in detail.

The above-mentioned IL-1β derivative has the same structure as the above a) to IL-β in the amino acid sequence represented by the above formula (A).
It is a polypeptide containing an amino acid sequence that satisfies one or more of the requirements of d). Preferred derivatives are those having an amino acid sequence that satisfies at least one of the requirements a) to c) and those having an amino acid sequence that satisfies at least one of the requirements a) to c) and d) at the same time. . Preferred specific examples of the polypeptide that is the IL-1β derivative are as follows.

1) A polypeptide in which at least the Ala position has been deleted or substituted with another amino acid residue.

2) A polypeptide in which at least Val in position 3 has been deleted or replaced with another amino acid residue.

3) A polypeptide wherein at least the 4-position Arg has been deleted or replaced with another amino acid residue.

4) A polypeptide wherein at least Ser at position 5 has been deleted or replaced with another amino acid residue.

5) A polypeptide wherein at least Cys at position 8 has been deleted or replaced with another amino acid residue.

6) A polypeptide wherein at least position 11 Arg has been deleted or replaced with another amino acid residue.

7) A polypeptide wherein at least His at position 30 has been deleted or replaced with another amino acid residue.

8) A polypeptide in which at least Cys at position 71 has been deleted or replaced with another amino acid residue.

9) A polypeptide wherein at least Lys at position 93 has been deleted or replaced with another amino acid residue.

10) A polypeptide wherein at least Lys at position 97 has been deleted or replaced with another amino acid residue.

11) A polypeptide wherein at least position 98 Arg has been deleted or replaced with another amino acid residue.

12) A polypeptide wherein at least Phe at position 99 has been deleted or substituted with another amino acid residue.

13) A polypeptide having at least Lys at position 103 deleted or substituted with another amino acid residue.

14) A polypeptide wherein at least Trp at position 120 has been deleted or replaced with another amino acid residue.

15) A polypeptide wherein at least Tyr at position 121 has been deleted or substituted with another amino acid residue.

16) A polypeptide wherein at least Ser at position 153 has been deleted or substituted with another amino acid residue.

17) At least the amino acid sequence from Ala at position 1 to Val at position 3 is deleted, the amino acid sequence from Ala at position 1 to Leu at position 6 or the amino acid sequence from Ala at position 1 to Thr at position 9 is at least deleted. Polypeptide.

18) Amino acid sequence from Val at position 151 to Ser at position 153, amino acid sequence from Gln at position 149 to Ser at position 153, amino acid sequence from Asp at position 145 to Ser at position 153, Ser from position Gln at position 141 to Ser at position 153 A polypeptide having at least the amino acid sequence, the amino acid sequence from Tyr 121 to Ser 153, or the amino acid sequence from Lys 103 to Ser 153.

19) A polypeptide having at least an amino acid residue at the N-terminal of the formula (A).

20) At the N-terminus of the formula (A), the amino acid sequence represented by the formula (B) is changed from Ala at position 112 'to Asp at position 116'.
Amino acid sequence from position 77 'Met to position 116' Asp
Amino acid sequence from Met 71 'to Asp 116'
Amino acid sequence leading to 32 'Met to 116' Ser
Or the amino acid sequence from 1 'Met to 116' Asp
A polypeptide having at least an amino acid sequence that leads to

The above IL-1β derivative comprises an amino acid sequence in which a specific amino acid residue at a specific position in the amino acid sequence of IL-1β is replaced with another amino acid residue and an amino acid sequence in which an amino acid residue is added at a specific position. The amino acid residue which can be substituted and added may be any residue of an α-amino acid constituting a human protein, and is preferably a neutral amino acid residue. . However, Cys may form an intramolecular or intermolecular disulfide bond based on its SH group, and in consideration of this, the amino acid residue is preferably the above-mentioned amino acid residue other than Cys. Particularly preferred is, for example, Gly, Lys, Gln or Asp in the case of 4-position Arg,
Ser or Ala for ys, Gln for 11th Arg
, Tyr for 30th His, and S for 71st Cys
er, Ala or Val, and in the case of Lys at position 93, Leu or Asp
98 for Arg, Leu for 103 Lys, Gln for 120 Trp, Arg for 121 Tyr, Gln for 121 Tyr, and Met for addition to the N-terminus. L
Eu, Arg or Asp can be exemplified.

The IL-1β derivative and IL-1β
Are, for example, LAF activity, tumor cell growth inhibitory activity (GIF
Activity), that is, the activity of specifically inhibiting the growth of tumor cells, a colony stimulating factor (Colony stimulating fac
tor: CSF), interferon (IF)
N), interleukin-2 (IL-
2), interleukin-3 (interleukin-3: IL-
3), etc., the activity of promoting the production of various cytokines, that is, the activity of, for example, acting on human cells to remarkably promote the production of those cytokines, the anti-inflammatory activity, particularly, for example, arthritis when administered to arthritis model animals. Activity to effectively suppress the progression of radiation, action to prevent radiation damage,
That is, it has an action of preventing or preventing whole body irradiation at the time of bone marrow transplantation, irradiation at the time of cancer treatment or the like, a biological disorder or a serious side effect at the time of a radiation accident, and the like. The above-mentioned IL-1β derivative is excellent in at least one of the above activities, or (and) is excellent in that it has lower toxicity and fewer side effects. Accordingly, the above-mentioned IL-1β derivative and IL-1β can be used as an immune system stimulant, for example, for promoting antibody production or enhancing the effect of a vaccine, an antitumor agent, for example, a cytokine production promoting agent such as CSF, IL-2, IL-3, etc. It is useful as a pharmaceutical such as an inflammatory agent or a radiation damage inhibitor.

In particular, a new finding that the above-mentioned IL-1β and its derivatives have a remarkable effect on inflammation such as arthritis has been reported.
Is surprising, according to the fact that has been implicated in mediating and causing inflammation. The above IL-
1β derivatives are superior in at least one of the above activities or / and are superior in that they are less toxic and have fewer side effects.

In particular, the above-mentioned IL-1β and its derivatives are effective as CSF production promoters. When these are administered to humans, there is no danger of virus infection, antigen-antibody reaction, etc. Granulopenia due to bone marrow hypoplasia after radiation therapy can be effectively restored (granulocytopenia therapeutic agent). The above CSF production promoter can also be effectively used as an agent for preventing and treating various diseases based on the action of CSF due to its original CSF production promotion action. For example, CSF has the effect of promoting the function of granulocytes and macrophages [Lopez, AF et al., J. Immm.
unol., 131, 2983 (1983), Handam et al. (Handam, E. et a
l., ibid., 122, 1134 (1979) and Vadas, MAet a.
130, 795 (1983)], and is expected to have clinical application as a preventive and therapeutic agent for various infectious diseases, and the above CSF production promoter is also expected to have clinical application.

In particular, a so-called opportunistic infection or terminal, in which a pathogen that has been harmless until now exhibits pathogenicity and is induced in an individual (compromised host) whose biological defense ability has been reduced or impaired in recent years. infecti
on), pathogens that cause clinical problems (causing bacteria) are Pseudomonas (Pseudomonas), Gram-negative bacilli such as Serratia, Herpes (Herpes simplex, HSV), Barricella zoster (Varicella zoster, VZV), Viruses such as cytomegalovirus (Cytomegalovirus, CMV), Candida (Candida albicans), Aspergillus (Aspergi
llus fumigatus), Nocardia asteroide
a) etc., protozoa such as Carinii protozoa (Pneumocystiscarinii), Toxoplasma (Toxoplasma gondii), etc., and currently used antibiotics are unlikely to exert a sufficient effect on such pathogens, and new anti-opportunistic infections Research and development of drugs are eagerly awaited. The IL-1β derivative is also useful as a prophylactic and therapeutic agent for such opportunistic infections, and in particular, for various infections during the administration of anticancer drugs where such opportunistic infections are frequently observed, ie, during chemotherapy or bone marrow transplantation for acute leukemia. It is useful as an agent for preventing and treating complications such as diseases such as gandidosis, cryptococcosis, aspergillosis, zygomycosis, black fungal infection, viral infection, cytomegalovirus pneumonia, and the like.

In addition to the above-mentioned pharmaceutical uses, IL-1β and its derivatives can be used extremely effectively, for example, in the production of various useful cytokines from cell lines in vitro based on their cytokine production promoting activity. Production of natural cytokines from such cell lines
In particular, attention has been paid to cytokines that are glycoproteins, and useful cytokines can be obtained efficiently and in large quantities.

In the above-mentioned IL-1β derivative, at least the Cys at position 71 is substituted or deleted, especially the above-mentioned Cys
Substituted with another amino acid residue, for example, Ser, Ala, Val, etc., shows high activity.

At least 4-position Arg, 93-position Lys,
The IL-1β derivative in which Cys at position 8 has been substituted or deleted, and the derivative in which at least one amino acid residue after position 103 has been deleted have weak prostaglandin E (PGE) production promoting effects. Therefore, a derivative having less side effects such as exothermic effect and toxicity, and further having a substitution or deletion of at least the 4-position Arg or the 93-position Lys, has a CSF production promoting activity and a lower activity than the GIF and LAF activities. Anti-inflammatory activity has stronger characteristics.

Further, in the above derivatives, those having at least a specific amino acid residue or polypeptide added to the N-terminus of the formula (A) have a higher C-amino acid activity than the GIF activity and the LAF activity.
It is more effective when used as a medicament, particularly as an oral preparation or a suppository, in that it has a higher SF production promoting action and an anti-inflammatory action, and has low toxicity and long-lasting action.

Further, the above derivatives, especially at least Cys at the 8-position
And / or those in which Cys at position 71 has been substituted or deleted, particularly those in which the above Cys has been substituted with another amino acid residue such as Ser, Ala, Val, etc., binds to the IL-1 receptor under various conditions. Excellent in properties.

Among the above-mentioned derivatives, those containing less or no Cys in the molecule as compared to IL-1β consider unnecessary formation of intramolecular or intermolecular bonds based on the SH group of Cys. It is more preferable.

The specific polypeptide described above, ie, IL-
1β and its derivatives can be produced, for example, by genetic engineering techniques. That is, it can be produced by utilizing a gene encoding the specific polypeptide, integrating the gene into a vector of a microorganism, and replicating, transcribing and translating in the cells of the microorganism. This method is particularly advantageous in that mass production is possible.

The gene used in the above method can be totally synthesized by chemical synthesis of nucleic acids according to a conventional method, for example, a conventional method such as the phosphite triester method (Nature, 310, 105 (1984)). Is IL-
It is convenient to synthesize using a gene encoding 1β or its precursor, for example, by modifying the gene into a nucleic acid sequence encoding the specific amino acid sequence according to a conventional method including the above-mentioned chemical synthesis means. Can be manufactured more easily.

The gene coding for IL-1β or its precursor is known, and we have also filed a previous application (Japanese Patent Application No. 60-13 / 1987).
No. 8281, JP-A-62-174022), a gene encoding IL-1β has been obtained, and IL-1β has been successfully obtained by genetic engineering using this gene.

The above nucleic acid (base) sequence modification operation may be performed according to a known method, and is carried out in accordance with the amino acid sequence of a target polypeptide [Genetic engineering techniques include, for example, Molecular Cloning Cold Spring Harbor Laborat
ory (1982)]. For example, conventional means such as various enzyme treatments such as restriction enzymes, DNA ligase, polynucleotide kinase, and DNA polymerase for the purpose of cutting, binding, and phosphorylating DNA can be employed, and these enzymes are easily available as commercial products. Available. The isolation and purification of the gene or nucleic acid in each operation may be performed according to a conventional method, for example, agarose electrophoresis. Further, the replication of the obtained gene may be performed according to a method using an ordinary vector, as described in part below. In addition, a DNA fragment encoding a desired amino acid sequence and a synthetic linker can be easily produced by the above-described chemical synthesis. In the above, the codon corresponding to the desired amino acid is known per se, and the selection may be arbitrary, for example, according to a conventional method in consideration of the codon usage frequency of the host to be used (Nucl.Acids.Res., 9, 43-74 (1981)
]. For example, the codon modification of the nucleic acid sequence can be performed, for example, in the usual manner using a site-specific mutagenesis (Site-specific mutagenesis) using a primer of about 15 to 30 mer comprising a synthetic oligonucleotide encoding the desired modification. -Specific Mutagenesis) (Proc.Natl.Acad.Sci.,
81 , 5662-5666 (1984)].

The desired gene obtained by the above method is
For example, a chemical modification method of Maxam-Gilbert [Maxam-Gilb
ert, Meth.Enzym., 65 , 499-560 (1980)] and the dideoxynucleotide chain termination method using M13 phage [Messing,
J.and Vieira, J., Gene, 1 9, by such 269-276 (1982)], either the determination of the base sequence and is may perform confirmation, is not limited to this or the like of a variety of methods well known in the art Can also be adopted.

Thus, a gene encoding the polypeptide having the specific amino acid sequence described above is provided (hereinafter, this gene is referred to as "target gene").

The specific polypeptide can be produced by using the above-mentioned target gene according to a known general gene recombination technique. More specifically, a recombinant DNA capable of expressing the target gene in a host cell may be prepared, introduced into a host cell, transformed, and the transformant may be cultured.

As the host cell, any of eukaryotes and prokaryotes can be used. The eukaryotic cells include:
The vertebrate cells include, for example, vertebrate cells, yeast cells, and the like. Examples of the vertebrate cells include Cos cells, which are monkey cells [Y.
l, 23 , 175-182 (1981)) and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (G. Urlaub and
LAChasin, Proc. Natl. Acad. Sci., USA, 77 , 4216-4220
(1980))] etc. are often used, but are not limited thereto.
As a vertebrate cell expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of the gene to be expressed, can be used. May be held. An example of such an expression vector is pSV2dhfr carrying the early promoter of SV40.
[S. Subramani, R. Mulligan and P. Berg, Mol. Cell.Bio
l., 1 (9), 854-864] and the like, but is not limited thereto.

As eukaryotic microorganisms, yeasts are generally used, and among them, yeasts belonging to the genus Saccharomyces can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as the yeast include pAM82 having a promoter for the acid phosphatase gene (A. Miyanohara et al., Proc. Natl. A
cad. Sci., USA, 80 , 1-5 (1983)].

Escherichia coli and Bacillus subtilis are generally used as prokaryotic hosts. For example, a plasmid vector that can be replicated in the host bacteria is used, and a target gene is expressed upstream of the gene so that the gene can be expressed in this vector. An expression plasmid to which a promoter and an SD (Shine and Dalgarno) base sequence, and further an ATG necessary for initiation of protein synthesis is added can be used. As Escherichia coli as the host bacteria,
Escherichia coli K12 strain or the like is often used, and the vector is generally pBR322.
Is often used, but is not limited thereto, and any of various known strains and vectors can be used. As the promoter, for example, tryptophan promoter, P _L promoter, lac promoter, lpp promoter and the like can be used, and in each case, the target gene can be expressed.

The use of the tryptophan promoter as an example will be described in detail. The vector pTM1 having the tryptophan promoter and the SD sequence is used as an expression vector.
[Now Text Man, Metabolism, Vol.22,289 (1985)]
The gene encoding the desired polypeptide to which ATG has been added may be ligated to the restriction enzyme ClaI site located downstream of the sequence, if necessary.

The present invention is not limited to the direct expression system, but may be a fusion protein expression system utilizing, for example, β-galactosidase or β-lactamase.

As a method for introducing the thus obtained expression vector into a host cell and transforming the same by a method generally used, for example, cells which are mainly in the logarithmic growth phase are collected, treated with CaCl ₂ and naturally treated with DNA. Can be adopted in such a state that the vector is easily taken in. In the above method, as is generally known, in order to further improve the transformation efficiency, MgCl _{2 is used.}
Alternatively, RbCl and RbCl can coexist in the medium.
Alternatively, a method of transforming a host cell into spheroplasts or protoplasts and then transforming the host cells can be employed.

The desired transformant thus obtained can be cultured according to a conventional method, and the desired polypeptide is produced and accumulated by the culture. The medium used for the culture may be any of various media commonly used for ordinary cell culture, and specific examples thereof include, for example, L medium, E medium,
Various carbon sources generally known to M9 medium and the like,
A medium to which a nitrogen source, inorganic salts, vitamins, and the like are added can be exemplified. When the above tryptophan promoter is used, culturing can be generally performed using, for example, an M9 minimum medium to which casamino acid is added so that the promoter works. At any time, an agent for enhancing the action of the tryptophan promoter such as indoleacrylic acid can be added.

Purification and isolation of the target polypeptide, that is, the specific polypeptide from the culture containing the active substance thus obtained can be carried out according to a conventional method. In extracting the polypeptide from the host, it is more preferable to use mild conditions such as an osmotic pressure method from the viewpoint of maintaining the higher-order structure.

The above-mentioned purification and isolation can be carried out, for example, according to various processing operations utilizing the physical and chemical properties of the polypeptide [for example, “Biochemical Database II”, pp1175-1259, 1st edition, 1st edition. 1st print, June 23, 1980,
(See Tokyo Chemical Co., Ltd.). Specific examples of the method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), liquid chromatography, centrifugation, electrophoresis, affinity chromatography, dialysis Combinations of methods, methods, etc. can be adopted.

More specifically, the above operation can be performed, for example, as follows. That is, first, the target polypeptide is partially purified from the culture supernatant in advance. This partial purification includes, for example, treatment using an organic solvent such as acetone, methanol, ethanol, propanol, or dimethylformamide (DMF) or an acid such as acetic acid, perchloric acid (PCA), or trichloroacetic acid (TCA) as a protein precipitant; , Sodium sulfate, sodium phosphate and the like, and / or ultrafiltration using a dialysis membrane, a flat membrane, a hollow fiber membrane or the like. The operations and conditions of each of these processes may be the same as those of the normal processes.

Next, the crude product obtained above is subjected to gel filtration to obtain a fraction in which the activity of the target substance is recognized. The gel filtration agent used here is not particularly limited, and for example, any of dextran gel, polyacrylamide gel, agarose gel, polyacrylamide-agarose gel, cellulose and the like can be used. Specific examples of these include Sephadex G type, LH type, Cephaloose type, Cefacryl type (above, Pharmacia), Cellulofine (Chitsuso), Biogel P type, and Biogel A type (Bio-Rad) ), Ultrogel (LKB), TSK-G
Type (Tosoh Corporation) can be exemplified.

For the polypeptide of interest, the active fraction obtained by the above gel filtration is subjected to, for example, affinity chromatography using a hydroxyapatite column, ion exchange column chromatography such as the DEAE method, the CM method, and the SP method, and chromatography. It can be further purified by subjecting it to a cashing method, reversed-phase high-performance liquid chromatography, or a combination of these operations, and can be isolated as a homogeneous substance.

The above-described chromatofocusing method can be carried out by various known methods. As a column, for example, PB
E94 (manufactured by Pharmacia), a starting buffer such as imidazole-hydrochloric acid, and an eluent such as Polybuffer 74 (manufactured by Pharmacia) -hydrochloric acid (pH
4.0) can be used.

The reversed-phase high-performance liquid chromatography is as follows:
For example C ₄ Haipoa reverse phase HPLC column - with (Bio Rad (Bio-Rad Laboratories)), etc., can be performed using acetonitrile, trifluoroacetic acid (TFA), a mixed solvent such as water, and this such as transfer agent .

Thus, the specific polypeptide as IL-1β and its derivative can be isolated and obtained.

Hereinafter, the composition stabilized by the method of the present invention will be described in detail.

The composition contains human serum albumin or, if necessary, a saccharide, a surfactant and the like, together with the IL-1β active substance including the above-mentioned IL-1β and derivatives thereof. Is a mandatory requirement.

In the above, the saccharide is not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose; sugar alcohols such as mannitol, inositol and xylitol; disaccharides such as sucrose, maltose and lactose; dextran; Polysaccharides such as hydroxypropyl starch can be used, and these can be used alone or in combination of two or more. Among them, sucrose, maltose, mannitol, inositol, dextran and the like are particularly preferable.

The surfactant is not particularly limited, and both ionic and nonionic surfactants can be used. Among them, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type,
Surfactants such as sorbitan monoacyl esters and fatty acid glycerides can be preferably used.

The amount of the above-mentioned saccharide added was as follows.
About 0.1 mg or more per μg, preferably about 1-1
It is appropriate that the amount is in the range of about 00 mg, and the amount of the surfactant added is about 0.00
About 01 mg or more, preferably about 0.001 to 0.1 m
It is appropriate to set the range to about g. The amount of human serum albumin added was about 0.0 per μg of IL-1β active substance.
It is appropriate that the range is about 01 mg or more, preferably about 0.01 to 10 mg.

The composition of the present invention may be the same as a general pharmaceutical composition of this kind except that the above-mentioned specific components must be blended, and other pharmacologically active ingredients or conventional pharmaceutical compositions may be used. The components and the like may be arbitrarily mixed, and the composition thus obtained is used in various medical applications as described above, for example, immunostimulants, antitumor agents, cytokines, etc. for enhancing antibody production and vaccine effect and treating immunodeficiency. It can be effectively applied to production-promoting agents, anti-inflammatory agents, radiation damage preventing agents, therapeutic agents for opportunistic infections, and the like.

In particular, as other components that can be added to the composition of the present invention, ordinary sulfur-containing reducing agents are preferable from the viewpoint of further increasing the stabilization of the IL-1β active substance. Specific examples of the sulfur-containing reducing agent include cysteine, N-acetylhomocysteine, thioctic acid, thioglycolic acid and salts thereof, thioethanolamine, thioglycerol, sodium thiosulfate, thiolactic acid, dithiothreitol, Preferred examples include relatively mild reducing agents such as glutathione,
These can be used alone or in combination of two or more. The addition amount of these compounds is not particularly limited.
It is appropriate to use about 0.001 mg or more, preferably about 0.01 to 10 mg per 1 μg of β-active substance (the total amount thereof when two or more kinds are used in combination).

The composition of the present invention is suitably made isotonic with a buffer to give a stable isotonic preparation. As the buffer used here, for example, typically, citric acid-
PH 4-8 of sodium citrate, citric acid-sodium phosphate, acetic acid-sodium acetate, citric acid-borax
Various buffers having a pH of preferably about 5 to 6 can be exemplified.

The composition of the present invention is prepared in the form of a pharmaceutical composition by, for example, mixing a pharmacologically effective amount of an IL-1β active substance and the above-mentioned specific ingredients with a suitable pharmaceutical carrier. As the pharmaceutical carrier, any excipient or diluent such as a filler, a bulking agent, a binder, a humectant, a disintegrant and the like which are usually used in the preparation of a pharmaceutical preparation according to the use form can be used. The form of the pharmaceutical composition is not particularly limited as long as it effectively contains the active ingredient IL-1β active substance, and may be a solid preparation such as tablets, powders, granules, and pills. It may be in the form of an injection, such as a solution, a suspension, or an emulsion. It can also be a dried product which can be made into a liquid form by adding an appropriate carrier before use. Any of these pharmaceutical compositions can be prepared according to a conventional method.

The obtained pharmaceutical preparation is administered by an appropriate administration route according to the form of the pharmaceutical composition, for example, a pharmaceutical preparation in the form of an injection is administered by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration and the like. Pharmaceutical preparations in solid form can be administered orally or enterally. The amount of the active ingredient in the pharmaceutical preparation and the dosage of the preparation are appropriately selected depending on the administration method of the preparation, the administration form, the purpose of use, the symptoms of the patient to whom the preparation is applied, etc. The active ingredient is prepared in the form of a preparation containing about 0.0000001 to 80% by weight, and the amount of the active ingredient contained in the preparation is about 0.000 per adult per day.
It is desirable to administer in the range of about 1 μg to 100 μg. The administration need not be once a day but can be divided into three to four times.

[0076]

According to the present invention, there is provided a stabilized composition of an active substance of IL-1β which has attracted attention and is expected as a medicine.

The composition of the present invention has excellent properties in terms of the stability of the active ingredient IL-1β, and can be prepared, for example, in a desired preparation as a usual pharmaceutical form such as a freeze treatment or a freeze-dry treatment. It is stable for a long period of time even under ordinary storage conditions of pharmaceuticals, and is extremely useful in such fields.

[0078]

The present invention will be described in more detail with reference to the following examples.

In each example, the active substance of IL-1β (IL-1β derivative) used as an active ingredient is indicated by the abbreviations shown in Table 1 below. Among them, polypeptides I to XXXXVIII are described in 63-1523
No. 98, the polypeptide XXXXI
X to XXXXXXVI are obtained by the following production reference examples according to the above. The measurement of IL-1 activity and GIF activity by these methods is according to the method described in the publication.

[0080]

[Table 1]

[0081]

[Table 2]

[0082]

[Table 3]

[0083]

[Table 4]

[0084]

[Table 5]

[0085]

Production Reference Example 1 Production Example 1 described in JP-A-63-152398
According to the above, each polypeptide (IL-1β derivative) shown in the following Table 2 was obtained by site-specific mutagenesis using the plasmid p trp GIF-α.

The expression of each polypeptide, the measurement and purification of GIF activity are in accordance with the method described in the above-mentioned publication, and SDS-PAGE is also referred to in Reference Example 2- (4).
The method was followed. The same applies to the following examples unless otherwise specified.

[0087]

[Table 6]

[0088]

[Table 7]

[0089]

[Table 8]

[0090]

[Production Reference Example 2] This is an example in which IL-1β derivatives (polypeptides XXXXXXIII to XXXXXXVI) in which various amino acid residues are added to the N-terminal of IL-1β are produced.

Near the N-terminal region, there is a ClaI site 5 bases upstream of the initiation codon ATG for protein synthesis, and an MspI site downstream of the ClaI site at the 11th Arg.
There is only a site, and when trying to insert a linker DNA between these two sites, the linker becomes too long, so that an appropriate restriction is first made at an amino acid closer to the N-terminus between these restriction enzyme sites. The creation of an enzyme site was examined, and as a result, the codon of the fourth Arg was changed from CGT to AGA, and Bgl of AGATCT was inserted between the next Ser.
II restriction enzyme site was created, and between the created BglII site and the ClaI site, various synthetic linkers containing a previously synthesized mutation region were replaced to construct a desired gene, which was expressed in E. coli, Was obtained. Hereinafter, the method will be described in detail.

(1) IL- having restriction enzyme BglII site
Preparation of 1β expression plasmid p trp GIF-α using the trp promoter: 4921
bp) was digested with the restriction enzymes ClaI and BamHI to give 57
Two fragment DNAs of 9 bp and 4342 bp were obtained.
The 579 bp fragment DNA was further digested with MspI to obtain a 543 bp fragment DNA having an MspI site at the 5 'end and a BamHI site at the 3' end. Next, a linker DNA having the following sequence was synthesized, and its 5 'end was phosphorylated using T4 polynucleotide kinase.

[0093]

[Table 9]

The phosphorylated linker was ligated to the above 543 bp fragment using T4 DNA ligase, and a DNA fragment having a ClaI site at the 5 ′ end, a BglII, MspI site at the middle and a BamHI site at the 3 ′ end ( 579 bp).

Finally, the 579 bp fragment obtained above and the 4342 bp fragment were combined with T4 DNA
By ligating with ligase, a new BglII
IL-1β expression plasmid having a site (ptrp IL-1
β, H2: 4921 bp).

The resulting plasmid was transformed into Escherichia coli HB101, and the resulting clone was subjected to DNA sequencing. As a result, it was confirmed that the gene was an IL-1β expression gene having a BglII site as intended.

(2) IL-1 by synthetic DNA linker
Preparation of β-terminal Mutein Addition Protein The plasmid DNA was replaced with a synthetic DNA linker and a vector DNA between the ClaI-BglII restriction enzyme sites of the plasmid p trp IL-1β, H2 obtained in (1) above.
A mutant protein having the desired amino acid added to the N-terminal of 1β was prepared.

The amino acid to be added is IL-1β
5 amino acid sequence derived from the precursor (Ala-Tyr-Val-His
-Asp; AYVHD), acidic amino acids (Asp; D), neutral amino acids (Leu; L) and basic amino acids (Arg;
R) one each. These displacement proteins were referred to as [AYVHD] -IL-1β and [D] -IL-
Abbreviated as 1β, [L] -IL-1β and [R] -IL-1β.

The synthetic linker DNA sequences are as follows.

Linker for adding AYVHD

[0101]

[Table 10]

Linker for adding D

[0103]

[Table 11]

Linker for adding L

[0105]

[Table 12]

Linker for adding R

[0107]

[Table 13]

First, the IL-β expression plasmid p trp IL-
1β, H2 was digested with ClaI and BglII to obtain 4.9 kb.
A fragment DNA of p was obtained. Next, the above linkers were synthesized, and their 5 'ends were phosphorylated using T4 polynucleotide kinase. Each of the resulting synthetic linkers having phosphorylated at the 5 ′ end, and 4.
The 9 kbp fragment DNA was ligated with T4 DNA ligase to construct four types of IL-1β N-terminal addition mutant protein expression plasmids.

Each of the plasmids was transformed into Escherichia coli HB101, and the resulting clone was subjected to DNA sequencing to confirm the target DNA sequence.

(3) Expression and Purification of IL-1β N-terminal Added Mutant Protein The HB101 strain (K12 strain) transformed with each of the expression plasmids obtained in (2) above was incubated at 37 ° C. in M9 medium.
With shaking.

The cells collected by centrifugation were treated with 1 M disodium hydrogen phosphate at 4 ° C., dialyzed against 5 mM Tris-HCl buffer (pH 8.0), and disrupted by osmotic shock. .

After adjusting the pH of the dialysate to about 4 with acetic acid,
The resulting precipitate was removed by centrifugation, and the supernatant was subjected to cation exchange high performance liquid chromatography (HPLC, using a SP-5PW column manufactured by Tosoh Corporation), and the main peak was further purified by re-chromatography. Each IL-1β mutein obtained by performing the above cation exchange HPLC twice was purified into a substantially single band by SDS-PAGE electrophoresis.

Further, ultrafiltration was performed using an Amicon YM-5 membrane, and the solvent was changed from acetate buffer (pH 5.5) to water.

The properties of each of the obtained mutant proteins were determined in the following 3rd.
These are summarized in the table.

[0115]

[Table 14]

The following activity tests were performed on IL-1β (polypeptide I) and its derivatives.

The LAF activity (U) per polypeptide I-equivalent protein amount (mg) measured by the RIA method using the above-mentioned antiserum against polypeptide I is as follows:
It is as shown in the table.

[0118]

[Table 15]

[0119]

[Pharmacological Test Example 1] Test for promoting effect of polypeptide I on CSF production Test for promoting effect on CSF production of human lung cells As a CSF-producing strain, a human lung cell-derived strain HFL-1 (Hu
The following tests were performed using man Embryonic lung Fibroblasts (ATCC registered cell line No. CCL-153).

First, a 10% fetal bovine serum-supplemented hamster 12K culture solution [Ham, RG, Proc. Natl. AcD> Sc was prepared so that the HFL-1 cells had a cell concentration of 2 × 10 ⁵ cells / ml.
i., 53 , 288 (1965)]. Next, the polypeptide I obtained in the above Reference Example prepared at various concentrations was added to the above cell suspension, and 37
At 24 ° C. for 24 hours, 48 hours and 72 hours, collecting the respective culture supernatants, and producing and accumulating CSF in the respective culture supernatants.
The amount was determined using mouse bone marrow cells (Lewis, IC
et al., J.Immunol., 1 28, 168 (1982) ].

FIG. 1 shows the results at each culture time (hr) obtained using the polypeptide I. In the figure, the horizontal axis indicates the concentration of polypeptide I (GIF unit / ml), and the vertical axis indicates CSF activity (unit / ml).

From the above results, it is clear that the addition of polypeptide I enhances the amount of CSF produced by the HFL-1 cell line by several hundred times compared to the absence of the polypeptide. .

Examination of Promoting Effect of Polypeptide I on CSF Production of Human Skin-Derived Cells CRL-1445 (AT
CC. No.), the following tests were conducted. That is, the above cells were adjusted to a cell concentration of 2 × 10 ⁵ cells / ml.
Dulbecco's MEM culture solution supplemented with 0% fetal bovine serum [Dulbeco,
R. and Freeman, G., Virology, 8, 396 (1959)]. Various concentrations of the polypeptide I obtained in Reference Example were added to the above cell suspension, and the suspension was incubated at 37 ° C. in a carbon dioxide incubator at 37 ° C.
After culturing for 4, 48 and 72 hours, the culture supernatant was collected, and the amount of CSF produced was measured using mouse bone marrow cells in the same manner as described above.

The obtained result was obtained in the same manner as in FIG.
Shown in the figure.

FIG. 2 shows that the GIF activity was 1 unit / m 2
It is clear that the addition of 1 or more polypeptides I to human normal skin-derived fibroblasts significantly enhances the ability of the cells to produce CSF.

Test of promoting effect on CSF production in vivo When polypeptide I was administered in vivo, C
The expression of the SF production enhancing effect was tested by the following animal experiments.

That is, a normal mouse (BALB / C mouse, purchased from Shizuoka Prefectural Experimental Animal Cooperative) was added to various amounts of the polypeptide I obtained in Reference Examples (10 ³ as GIF activity).
The 10 ⁵ units / individual) were intravenously administered. Blood was collected from each experimental animal at 2, 4, 8, 12 and 24 hours after the administration, and the CSF concentration in the serum was measured using mouse bone marrow cells.

The results are shown in FIG. In the figure, the horizontal axis indicates the time (hr) after administration of polypeptide I at various concentrations (GIF units / individual), and the vertical axis indicates CSF activity (unit / ml serum). (1) shows 100,000 G of polypeptide I.
IF unit / individual administration group; (2) 10,000 GIF unit /
The individual administration group, (3) shows the same 1000 GIF unit / individual administration group, and (4) shows the control group (HSA 10 μg / individual administration group).

From FIG. 3, it was found that when polypeptide I was given to animals, the CSF concentration in the serum of the animals was extremely high. In other words, it was confirmed that polypeptide I had an effect of significantly increasing CSF production in vivo in proportion to the injected amount.

[0130]

[Pharmacological test example 2] Anti-arthritic test of polypeptide I [Pearson, CM, Proc. Soc. Exp. Biol. Me
d., 91 , 95 (1956)) and Ward and Jones (Ward, J.
R., Jones, RS, Arthritis Rheumatism, 5, 557 (1962)
], Adjuvant arthritis rats were prepared. That is, female S. D. Mycobacterium butyricu was found in the skin of the ridge of the rat.
m) 0.05 ml of adjuvant with dead bacteria suspended in liquid paraffin was injected. On the 14th day, the animals were divided into groups based on foot swelling (n = 6), and from the next day, the polypeptide I or its solvent (saline; control group) was intradermally administered for 5 days. The effect on arthritis was assessed by measuring paw volume daily.

The results are shown in FIG. In the figure, the horizontal axis represents days (days) after adjuvant administration, and the vertical axis represents paw volume (× 0.
01 ml). In the figure, (1) shows a group administered with 100,000 GIF units / individual of polypeptide I, (2) shows a group administered with 10,000 GIF units / individual, and (3) shows a group given with 1000 GIF.
Unit / individual administration group, (4) 100 GIF unit / individual administration group, (5) control group (physiological saline administration group),
(6) shows a normal rat group.

FIG. 4 shows that the foot swelling of the control group (graph (5)) was exacerbated until day 23, whereas the polypeptide I
In the administration groups (Graphs (1) to (4)), an inhibitory effect on foot swelling was observed on the fourth day (18 days after adjuvant administration), and 4 days after the last administration (adjuvant administration) 23 days later), it was confirmed that the progression of arthritis could be prevented.

[0133]

[Pharmacological Test Example 3] Test of effect of promoting IL-1β derivative on CSF production Cell line U-373MG [ATCC HTB17, Gliob
lastoma, Astrocytoma, Human], the following test was conducted.

[0134] The above cells were adjusted to a cell concentration of 2 x 10 ⁵ cells / ml with 10% FCS (GIBCO) and MEM.
Suspended in Eagle MEM medium (Nissui) supplemented with non-essential amino acids (Flow) and MEM sodium pyruvate (Flow), test substances were added at various concentrations, and carbon dioxide gas culture was performed. The cells were cultured at 37 ° C. for 24 hours in a vessel.

Each culture supernatant was collected, and the amount of CSF produced and accumulated in the culture supernatant was measured using mouse bone marrow cells [Lewis, IC et al., J. Immunol., 128, 168]. (19
2)].

The results are shown in FIG. In the figure, the horizontal axis represents the concentration of the test substance (ng / ml), and the vertical axis represents the CSF activity (U
/ Ml). Curves (1) to (7) in the figure show the results when the following polypeptides were used as test substances.

Curve (1): Polypeptide VI curve (2) Polypeptide II curve (3) Polypeptide VIII curve (4) Polypeptide V curve (5) Polypeptide IV curve (6) Polypeptide III Curve (7): Polypeptide XXX

[0138]

[Pharmacological Test Example 4] Anti-inflammatory test of IL-1β derivative The method of Winter et al. [Proc. Soc. Exptl. Bio
Med., 111, 544-547 (1962)].

That is, 6-8 week old male rats (Spraque Da
(Wley strain, Charles River Japan) was used in groups of 6 to 8 animals per group based on body weight on the day before the experiment. Using carrageenin (manufactured by Marine Colloid) as a proinflammatory agent suspended in physiological saline at a concentration of 1%, 0.1 ml was injected subcutaneously into the right hind footpad of rats to induce paw edema. I let it. To assess paw edema, the right hind footpad volume was measured with a plethysmometer (plethy
smometer, Ugo-Vasile). The edema rate (volume increase rate after injection of the proinflammatory agent with respect to the
%).

The test substance was dissolved and diluted in Dulbecco's phosphate buffered saline, and 0.1 ml was injected into the back skin of the rat one hour before injection of the proinflammatory agent. A solvent-administered group was prepared as a control group and subjected to the same experiment.

The results are shown in FIG.

In the figure, the horizontal axis represents the time (h)
r), and the vertical axis indicates the edema rate (%). In the figure, curve (1) represents the control group, curve (2) represents the group administered with 0.1 μg of polypeptide VI, curve (3) represents the group administered with 1 μg of polypeptide VI, and curve (4) represents the group 10 of peptide VI
The μg administration group is shown.

[0143]

[Pharmacological Test Example 5] Test for prevention of radiation injury by IL-1β derivative 1 μg / mouse of polypeptide VI or 0.2 hour before irradiation of BALB / c mouse (9 weeks old) with lethal dose of X-ray. 3 μg / mouse was injected intraperitoneally. Using an X-ray irradiator (MBR-1505R, Hitachi Medical)
X-rays of 850 X-rays are irradiated to the whole body of the mouse,
Thereafter, its survival was confirmed every day. In addition, a PBS administration group was set as a control.

The results are shown in FIG. In the figure, the horizontal axis is X
The number of days after irradiation (day), the ordinate indicates the survival rate (%) of the test animal, and the curve (1) indicates 1 μg of the polypeptide VI.
Curve (2) shows 0.3 μg of polypeptide VI.
The administration group and the curve (3) represent the control group, respectively.

From FIG. 7, it was found that in the control group, all the animals died on the 18th day after X-ray irradiation, whereas the polypeptide VI
In the administration group, the effect of preventing radiation damage was observed depending on the dose, and it was confirmed that about 80% of the 1 μg administration group was prevented from death due to radiation damage and survived.

[0146]

[Pharmacological test example 6] Test for protective effect of opportunistic infection of IL-1β derivative The following test was carried out using an easily infected model mouse.

Male ICR mice (6 weeks old) were used as test animals (7 mice per group), and on the first day, 100 mg / kg of 5-fluorouracil (5-Fu, manufactured by Kyowa Hakko) was administered intravenously. . On days 2, 4, and 6, 1 μg / mouse of polypeptide VI was administered subcutaneously, and on day 7,
A predetermined amount of Pseudomonas aeruginosa E-2 was intraperitoneally administered to infect. On the 10th day, the number of surviving test animals was counted to determine the survival rate (%).

The results are shown in FIGS. 8 (1) to (3).

FIG. 8 (1) shows the results of the above experimental group, and FIG. 8 (2) shows that the polypeptide VI was not administered (5-Fu).
(3) was the same as 5-
The results of the control group to which neither Fu nor polypeptide VI was administered are shown.

In FIG. 8, the ordinate represents the survival rate (%), and the abscissa represents the groups A to E employing the following doses of Pseudomonas aeruginosa.

Group A: 19000 cells / mouse administered group B Group: 3800 cells / mouse administered group C: 750 cells / mouse administered group D: 150 cells / mouse administered group E group: 30 cells / Mouse administration group F group: 6 bacterial counts / mouse administration group <Method for producing cytokine from animal cells> Various concentrations of polypeptides XXXVII and 0.0
In the presence of 1% PHA-P, HBS-2C5B2 cells [J.
Immunol., 131, 1682-1689 (1985)] was cultured at 2 × 10 ⁵ cells / well. The supernatant after 24 hours of culture was collected, and its IL-2 activity was measured according to the method of KASmith et al. For IL-2 dependent mouse T cells (CTLL2).
[J. Immunol., 120, 2027 (1978)].

The results are shown in Table 5 below.

[0153]

[Table 16]

U-373MG cells were transformed with 10% FCS
Culture to confluency in supplemented RPMI-1640 medium and add 2 more
Incubation for 18 hours in the above medium with or without 0 ng / ml of the polypeptide XXXVII (control).

After removing the medium, RNA was extracted by the guanidinium / cesium chloride method, and oligo (dT) was extracted.
-Poly (A) by cellulose chromatography
^* RNA (mRNA) was obtained. According to Northern blotting, the above poly (A)
^* 10 μg of RNA was subjected to agarose gel (1.2%) electrophoresis, fractionated, and then transferred to a nitrocellulose filter. Baking at 80 ° C under reduced pressure, 20 mM Tris H
After treatment in Cl (pH 8.0) at 100 ° C. for 5 minutes,
50% formamide, 5 × ssc, 50 mM sodium phosphate (pH 6.5), 4 × denhardt solution (De
nhardt's solution) and 200 μg / ml denatured Salmon Speram DNA at 42 ° C. for prehybridization. Five hours later, GM-CSF cDNA radiolabeled by nick translation [Scienc
e, 228 , 810 (1985)] or the PstI-NcoI DNA fragment or B
KpnI- of SF-2 cDNA [Nature, 324, 73 (1986)]
Hybridization was performed at 42 ° C. for 20 hours with the BamHI DNA fragment. Filter with 0.1% SDS
Add 2xssc at room temperature for 15 minutes, 0.1% SDS
Washing was performed at 50 ° C. for 1 hour with additional 0.1 × ssc. Autoradiogram was performed overnight at -70 ° C using an intensifying screen.

As a result, both when the GM-CSF DNA fragment was used and when the BSF-2 DNA fragment was used,
It has been found that the production of natural cytokines from animal cells can be performed efficiently by using the IL-1β derivative.

In applying the IL-1β derivative to such a method, it is sufficient to use an extremely small amount, usually about 10 ng / ml, and also facilitates the purification process of the induced cytokines.

When producing cytokines from animal cells, the IL-1β activator used to induce production is structurally stable under the conditions and IL-1β on the cell surface
Binding to the receptor is essential. That is, IL-1β
It is important that the activator binds to the IL-1 receptor and conveys the signals necessary for cytokine production into cells. Thus, the following test was performed for binding to the IL-1 receptor on fibroblasts. That is, Balb / 3T3 cells (clone A31: ATCC, CCL-163, 1 ×
To 10 ⁶ cells / well, 50,000 cpm / well of ¹²⁵ I-labeled polypeptide I (IL-1β) and 20 ng / ml of polypeptide I previously incubated at 37 ° C. in D-MEM with 10% FCS were added. The reaction was performed at 4 ° C. Remove the reaction solution with a Pasteur pipette and remove 10%
1 ml of FCS-added D-MEM was added, and the mixture was gently washed and the supernatant was discarded. After repeating this washing operation twice, 1 ml of 1%
The cells were solubilized with SDS and 0.2N NaOH, and the radioactivity (bound radioactivity) in the lysate and the lysate from which the wells were further washed were measured with a γ-counter.

The ¹²⁵ I-labeled polypeptide I was obtained by the method of Bolton and Hunter [Biochem.
J., 13 3, 529 (1973 ) was produced purified according] (specific activity: 2
50 μCi / μg protein or more).

The results obtained are shown in Table 6 below.

[0161]

[Table 17]

The stopping power in the table was determined by the following equation.

Stopping power (%) = (AC) / (AB) × 100 A: radioactivity bound in the absence of unlabeled polypeptide I B: radioactivity non-specifically adsorbed to the plate C: Actual value of bound radioactivity Such an index indicates the binding ability of coexisting polypeptide I to IL-1 receptor.

From Table 6 above, it can be seen that polypeptide I, ie, I
It has been clarified that L-1β itself decreases in binding force to the IL-1 receptor with time under cytokine-induced conditions.

In the above, polypeptide I and polypeptide VI which had been subjected to a 24 hour pre-incubation
Alternatively, the results of the same test using polypeptide XXXVII are shown in Table 7 in the same manner as in Table 6.

[0166]

[Table 18]

From the above results, it can be seen that in the production of cytokines from animal cells, the use of an IL-1β derivative is more preferable.

[0168]

Example 1 GIF activity [In the above-described method, A375S1 strain was used as human melanoma cells
45000 units / ml
(Approximately 1 μg protein / ml) of polypeptide VI, Tween 80 (Polysorbate 80: manufactured by Nippon Surfactant) 0.01 mg / ml and maltose 15 mg / m
l of each component was added to a 0.01 M citric acid-sodium citrate buffer (pH 6.0) and mixed, and the mixture was filtered (using a 0.22 μm membrane filter).
Thereafter, 1 ml of the filtrate was aseptically dispensed into vials and freeze-dried to prepare the composition of the present invention in the form of a preparation for injection. The preparation is used by dissolving it in 1 ml of physiological saline before use.

As described above, polypeptide I
The composition of the present invention in the form of a preparation for injection containing I or polypeptide XXV as an active ingredient was prepared.

[0170] Other IL-1β active substances can be prepared in substantially the same manner as injectable preparations.

Stability Test Using the preparation obtained above, its stability was tested by the following method.

(I) Method I: The above freeze-dried preparation was
Each was stored at 4 ° C. or room temperature in a glass bottle (airtight, light-shielded) container. The stability was evaluated after storage for 1, 2, 3, and 6 months, respectively, based on the following criteria.

<Judgment Criteria> Properties (appearance): When there was a white mass as in the start of the test, it was judged as “no change”.

Properties (dissolved): When the solution dissolved in 1 ml of physiological saline was colorless and transparent, it was judged as "no change".

Hydrogen ion concentration: pH value at start (5.6
The range of ± 0.2 of 5) was determined as “no change”.

Osmotic pressure ratio: When the value at the start was 100%, when the value was in the range of 95 to 105%, it was judged as "no change".

[0177] The above evaluation was made by averaging the results of three vials per sample.

(II) Results: At all storage temperatures and all storage periods, all the preparations of the present invention were judged as “no change” by the above criteria. Table 8 shows the measured data (average value) after storage for 6 months in the above test.

[0179]

[Table 19]

(III) Method II: The above freeze-dried preparation was dissolved in 1 ml of physiological saline, and then stored at 4 ° C. or at room temperature for 3 days. Was measured and evaluated.

(IV) Results: The results of the above Test II were
The results are shown in Table 9 in the same manner as in the table.

[0182]

[Table 20]

The results of the above tests (Tables 8 and 9) show that the composition of the present invention is extremely stable.

(V) Method III: The above freeze-dried preparation was stored at 4 ° C. or room temperature in a glass bottle (airtight, light-shielded) container. The stability was evaluated after storage for 1, 2 and 4 weeks based on the following criteria.

<Judgment Criteria> Content (%): The content of IL-1β active substance after the start of the test was 1
When it was set to 00%, when it was within the range of 95 to 105%, it was judged as "no change".

The content of the IL-1β active substance was measured by high performance liquid chromatography (HPLC: HPLC system manufactured by Tosoh Corporation) under the following conditions.

Column: TSK ODS-120T (4.
Solvent: Solution A = 0.1% TFA-water Solution B = 0.1% TFA-acetonitrile gradient program Detection: UV absorption (220 nm) (VI) Result: All were judged as "no change" at all storage temperatures and periods. The measured data (average) after storage for 4 weeks is shown in Table 10 below.

[0188]

[Table 21]

[0189]

Example 2 The composition of the present invention was prepared in the form of a preparation for injection, in the same manner as in Example 1, except that the components thereof were changed to the following I to E.

A. Polypeptide VI 10 μg / ml Tween 80 0.01 mg / ml Dextran 40 15 mg / ml cysteine 0.1 mg / ml HSA 1 mg / ml (using the same buffer) b. Polypeptide VI 10 μg / ml Tween 80 0.01 mg / ml sucrose 15 mg / ml cysteine 0.1 mg / ml (using the same buffer) c. Polypeptide VI 10 μg / ml Tween 80 0.01 mg / ml mannitol 15 mg / ml cysteine 0.1 mg / ml HSA 1 mg / ml (using the same buffer) d. B. Polypeptide VI 10 μg / ml Tween 80 0.01 mg / ml inositol 15 mg / ml cysteine 0.1 mg / ml HSA 1 mg / ml (using the same buffer) Polypeptide VI 10 μg / ml Tween 80 0.01 mg / ml Maltose 15 mg / ml Cysteine 0.1 mg / ml (0.01 M citrate-sodium citrate buffer (p
H5.0) Use) The stability of each preparation prepared above was tested by the following method.

(I) Method: The above preparation was stored in a glass bottle (airtight, light-shielded) container at 4 ° C. or 25 ° C., respectively, and after storage for 1, 2 and 4 weeks, the criteria shown in the above-mentioned stability test. According to the above, the properties (appearance and solution) and the content (%) of each preparation were evaluated to examine the stability.

(II) Results: All preparations (A to E above)
Was determined to be "no change" under all storage conditions. The measured data (average) after storage for 4 weeks is shown in Table 11 below.

[0193]

[Table 22]

From the above test results, the composition of the present invention was
It turns out that it is extremely excellent in stabilizing the IL-1β active substance.

[0195]

Example 3 IL-1β active substance [polypeptide VI] 0.1 μg
/ Ml or 0.01 μg / ml of the composition of the present invention having the following composition was placed in each of a glass vial, a silicon-coated glass vial, a polypropylene container and a polystyrene container to prepare the composition of the present invention.

<Combined composition> IL-1β active 0.1 or 0.01 μg / ml 0.01 M citrate buffer (pH 6.0) sucrose 5 mg cysteine 0.1 mg human serum albumin 0.01-10 mg / ml For each of the inventive compositions,
The following adsorption prevention test was performed. That is, after leaving each composition sample at 4 ° C. for 2 days and 4 days, respectively, IL-1
The remaining amount of the active substance was measured by the following enzyme immunoassay.

<Enzyme Immunoassay> A mouse anti-IL-1β activity monoclonal antibody (100 μl / well) is added to a 96-well microplate, and the mixture is left at 4 ° C. overnight. After washing, 400 μl of 1% bovine serum albumin is added, and left at room temperature for 30 minutes to perform blocking. After washing, add 100 μl of serial dilution of the sample,
After washing overnight, wash. Rabbit anti-IL-1β antibody [Cl
inica Chimica Acta, vol.166, p237-246 (1987) and Eu
rop. J. Immunolog., vol. 17, 1527-1530 (1987)]
Add 00 μl and leave at 37 ° C. for 2 hours. After washing, 100 μl of a solution of a peroxidase-labeled antibody rabbit globulin (manufactured by Bio-Rad) is added, and the mixture is left at 37 ° C. for 2 hours. After washing, 100 μl of the substrate solution is added and left at room temperature for 2 to 15 minutes. Stop the reaction with 2N sulfuric acid,
Measure the absorbance at 92 nm. Separately, a calibration curve is prepared using a solution having a known concentration of the IL-1β active substance, and the concentration of the IL-1β active substance in the sample is determined from the calibration curve.

Table 12 shows the obtained results.

However, in Table 12, 1 in the column of container type ^* indicates a glass vial, 2 indicates a silicon-coated glass vial, 3 indicates a polypropylene container, and 4 indicates a polystyrene container.

It is apparent from Table 12 that none of the composition samples of the present invention adsorb IL-1β to the container.

[0201]

[Table 23]

[0202]

[Table 24]

[0203]

Example 4 Human serum albumin (0.1 mg), 0.01 M citric acid-sodium citrate buffer (pH 6.0) and each component shown in Table 13 below were added to 1 μg of an IL-1β active substance (polypeptide VI). Add and mix, filter the mixture (0.22
After use of a μm membrane filter), the filtrate is aseptically removed
Each ml was dispensed into a glass vial and freeze-dried to prepare a composition of the present invention in the form of a formulation for injection.

[0204]

[Table 25]

The stability of each of the samples of the present invention prepared above was examined by the following tests. That is, each freeze-dried sample was placed in a glass vial (airtight, light-shielded) at 25 ° C., 50 ° C.
The cells were stored at a temperature of 70 ° C or 70 ° C for 1, 2 or 4 weeks, respectively (however, stored at 70 ° C for only 1 week).

The remaining amount of the active substance of IL-1β was measured by the following chromatography (HPLC: HPLC system manufactured by Tosoh Corporation).

Column: TSK C ₁₈ -NPR (4.6φ)
× 35 mm manufactured by Tosoh Corporation) Solvent: A solution = 0.1% TFA-water B solution = 0.1% TFA-acetonitrile gradient program Detection: UV absorption (210 nm) Residual rate (%) = (residual amount after elapse of predetermined time) / (initial amount) × 100 The results obtained are shown in Table 14.

[0208]

[Table 26]

As shown in Table 14 above, 4 weeks at 25 ° C.
No change was observed in all of the composition samples of the present invention when the sample was stored at 50 ° C. for 4 weeks.
No change was observed in the sample and the composition of the present invention.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of a CSF production promoting effect test of polypeptide I.

FIG. 2 is a graph showing the results of a CSF production promoting effect test of polypeptide I.

FIG. 3 is a graph showing the results of a CSF production promoting effect test of polypeptide I.

FIG. 4 is a graph showing the results of an anti-arthritis test of polypeptide I.

FIG. 5 is a graph showing the results of a CSF production promotion test of an IL-1β derivative.

FIG. 6 is a graph showing an anti-inflammatory test result of an IL-1β derivative.

FIG. 7 is a graph showing the results of a radiation damage prevention effect test of an IL-1β derivative.

FIG. 8 is a graph showing the results of an opportunistic infection prevention effect test of an IL-1β derivative.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-138222 (JP, A) JP-A-60-215631 (JP, A) JP-A-61-197527 (JP, A) JP-A-62 223129 (JP, A) JP-A-62-292726 (JP, A) JP-A-62-164631 (JP, A) International publication 88/969 (WO, A1) European publication 237073 (EP, A1) Medical Immunology, Vol. 12, No. 6 (1986) P. 753-760 (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 38/20

Claims (2)

(57) [Claims] 1. An interleukin-1β active substance,
Lyophilized , characterized by containing human serum albumin
A method for stabilizing an interleukin-1β composition in a dry form . 2. The method according to claim 1, wherein the interleukin-1β activity is human interleukin-1β.