JPH0611707B2 - Preventive agent for secondary illness and recovery accelerator - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention is secondary hypoadrenal function (adrenal atrophy), that is, it is caused or caused by administration of steroid hormones or after Cushing's syndrome. Preventive agent for secondary adrenal insufficiency and recovery accelerator, more specifically human interleukin 1β
The present invention relates to a preventive and recovery promoting agent for secondary adrenal insufficiency, which comprises (IL-1β) or a derivative thereof.

Conventional technology and its problems Due to recent advances in neurosurgery and endocrinology, Cushing's syndrome, which is a hyperglycosteroid overexpression from the adrenal cortex, can be completely cured by surgical treatment. Corticotropin of the anterior pituitary gland (A
Evolution of CTH, Adrenocorticotropic hormone) -producing tumors or adrenal glycosteroid-producing tumors leads to secondary adrenal insufficiency or insufficiency in most cases from 6 months to several years after surgery. That is, the corticotropin-releasing hormone released from the brain toward the pituitary gland by the secretion of a large amount of glucocorticoids, mainly cortisol, up to that time.
one; CRH) and the action of pituitary ACTH-secreting cells (the ACTH-secreting cells in the non-tumor tissue compressed around the tumor in ACTH-producing tumors) that are the original cortisol-secreting hormones However, even after the tumor is extirpated, its function cannot be restored for a long time. As a result, insufficiency of ACTH secretion from the pituitary gland occurs and persists, secretion of cortisol and adrenal androgens decreases, and the response of ACTH and cortisol to stress becomes less than normal. In addition, persistent reduction of ACTH basal secretion leads to atrophy of the adrenal cortex fasciculata and reticularis, which further reduces basal cortisol secretion and impairs the entire pituitary-adrenal system.

On the other hand, currently, for the treatment of autoimmune diseases, allergic diseases, shock, etc., steroid hormone agents (corticosteroid agents), especially hydrocortisone [(Hydrocortisone)]
Alternatively, various glucocorticoids synthesized based on [cortisol] are widely used in the clinical field.

However, these exogenous glucocorticoids often require long-term administration, which clinically leads to a metabolic state similar to Cushing's syndrome (iatrogenic Cushing's syndrome) and, conversely, in terms of endocrine function, Cushing's syndrome surgery is performed. Later, similar ACTH production and secretion is suppressed in the anterior pituitary gland, and due to this, secondary adverse adrenal function or failure is caused as a serious side effect.

In order to avoid the appearance of secondary adrenal insufficiency or failure as described above, it is considered that the dose of the adrenal cortical slaloid drug is once reduced or discontinued, but depending on the severity of the applied disease, the above-mentioned In some cases, it is impossible to discontinue drug administration.In such cases, the dose should be carefully reduced or switched to another corticosteroid and gradually reduced, and once persistent hypoadrenal function or failure appears,
If the administration of corticosteroids is suddenly stopped, there is a risk of shock death due to adrenocortical failure. In addition, the secondary adrenal insufficiency or failure that has once appeared may take several months or longer for the recovery to normalize even after discontinuation of steroid administration, and during that time, the therapeutic effect on the underlying disease due to steroid administration is expected. Instead, it causes serious difficulties in performing the treatment.

Currently, in the treatment of hypopituitarism, compensation therapy is performed to supplement hormones of the adrenal gland in diseases such as the brain (hypothalamus), pituitary gland, and peripheral hormone organs depending on the lesion site.
However, for persistent adrenal insufficiency or insufficiency, as is clear from the reasons described above, it is not the compensation by the hypothalamus or pituitary hormone which is the original primary lesion site, but the administration of a steroid agent that is a peripheral organ hormone. It is being implemented as a measure for improvement. This is because the ACTH administration therapy attempted so far stimulates the primary secretion of steroid hormones but does not lead to the general recovery of atrophic adrenal function, and the administration of CRH also results in the current CRH. This is because the effect is unknown in the drug product.

Therefore, the current treatment for persistent adrenal insufficiency or failure is
While supplementing the immediate hormone compensation with steroids, the overall function of the pituitary-adrenal system is merely passively awaiting its natural recovery.

Means for Solving the Problems In view of the above-mentioned present situation, the present applicant can prevent the occurrence of persistent adrenal insufficiency after Cushing's syndrome or the occurrence of secondary adrenal insufficiency or insufficiency caused by steroid administration, In addition, intensive research has been conducted for the purpose of providing a new drug capable of recovering the dysfunction after the occurrence. In the process, human IL-1β and its derivatives
It is completely different from the action of activating lymphocytes, which is its original biological activity, to enhance the production of interleukin 2 (IL-2) and the like and the production of antibodies, anti-inflammatory activity, radiation damage prevention action, etc. We discovered the fact that it has an effect of preventing or recovering from secondary adrenal insufficiency or insufficiency caused by Cushing's syndrome or by administration of steroids, thus preventing and recovering the above-mentioned secondary adrenal insufficiency or insufficiency as a new drug. We have established a promoter.

That is, the present invention relates to a preventive and recovery-promoting agent for secondary adrenal hypofunction, which comprises at least one selected from human IL-1β and its derivatives.

As described above, the drug of the present invention essentially requires at least one of human IL-1β and its derivatives as an active ingredient. The human IL-1β is the LAF (Lymphocyte Act)
a polypeptide having a 153 amino acid sequence represented by the following formula (1) having ivating factor activity [natural IL-1β; Proc. Natl. Acad. Sci., Vol. 81, 7907-7911 (1
984); Nature, Vol.315, 641 (1985); Nucleic Acid Researc
h, Vol. 13 (16) 5869 (1985), etc.], which may be isolated and purified from the cell of origin according to a conventional method, using mRNA of the cell of origin described above. Alternatively, it may be produced and isolated and purified by a microorganism such as Escherichia coli by a genetic engineering method.

Formula (1): Ala-Pro-Val-Arg-Ser-Leu-Asn-Cys-Thr-Leu-Arg-Asp-Se
r-Gln-Gln-Lys-Ser-Leu-Val-Met-Ser-Gly-Pro-Tyr-Glu-
Leu-Lys-Ala-Leu-His-Leu-Gln-Gly-Gln-Asp-Met-Glu-Gl
n-Gln-Val-Val-Phe-Ser-Met-Ser-Phe-Val-Gln-Gly-Glu-
Glu-Ser-Asn-Asp-Lys-Ile-Pro-Val-Ala-Leu-Gly-Leu-Ly
s-Glu-Lys-Asn-Leu-Tyr-Leu-Ser-Cys-Val-Leu-Lys-Asp-
Asp-Lys-Pro-Thr-Leu-Gln-Leu-Glu-Ser-Val-Asp-Pro-Ly
s-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe-Val-
Phe-Asn-Lys-Ile-Glu-Ile-Asn-Asn-Lys-Leu-Glu-Phe-Gl
u-Ser-Ala-Gln-Phe-Pro-Asn-Trp-Tyr-Ile-Ser-Thr-Ser-
Gln-Ala-Glu-Asn-Met-Pro-Val-Phe-Leu-Gly-Gly-Thr-Ly
s-Gly-Gly-Gln-Asp-Ile-Thr-Asp-Phe-Thr-Met-Gln-Phe-
Val-Ser-Ser Further, the IL-1β derivative includes a polypeptide having an amino acid sequence in which a part of the amino acid sequence of the natural IL-1β is modified, and a typical example thereof is the applicant's prior Can be exemplified by various derivatives related to the application [Japanese Patent Application Laid-Open No. 63-152398 (see EP Publication No. 0237967). Specific examples thereof include the amino acid sequence of IL-1β represented by the above formula (1). (a) 1st ^Ala , 3rd ^Val , 4th ^Arg , 5th ^Ser , 8th ^Cys , 1
1st ^Arg , 30th ^His , 71st ^Cys , 93rd ^Lys , 97th
^Lys , 98th ^Arg , 99th ^Phe , 103th ^Lys , 120th
^At least one amino acid residue selected from ^Trp , 121-position ^Tyr, and 153-position ^Ser is deleted or substituted with another amino acid residue; (b) ^{Ala at} position 1 to ^{Thr at} position 9; Or at least one amino acid residue therein is deleted (provided that 1-position ^Ala , 3-position ^Val , 4-position ^Arg described in (a) above).
(Except when at least one amino acid residue selected from the group consisting of ^{Ser at} position 5 and ^{Cys 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 has been deleted (however, 103th ^Lys and 120th position described in (a) above).
Except when at least one of the amino acid residues selected from the group consisting of ^Trp , 121-position ^Tyr and 153-position ^Ser is deleted), (d) the amino acid residue at the N-terminal of the formula (1) or The addition of an amino acid sequence from the 1'position ^Met to the 116 'position ^{Asp represented} by formula (2) or a partial amino acid sequence on the C-terminal side thereof, formula (2) Met-Ala-Glu-Val-Pro -Glu-Leu-Ala-Ser-Glu-Met-Met-Al
a-Tyr-Tyr-Ser-Gly-Asn-Glu-Asp-Asp-Leu-Phe-Phe-Glu-
Ala-Asp-Gly-Pro-Lys-Gln-Met-Lys-Cys-Ser-Phe-Gln-As
p-Leu-Asp-Leu-Cys-Pro-Leu-Asp-Gly-Gly-Ile-Gln-Leu-
Arg-Ile-Ser-Asp-His-His-Tyr-Ser-Lys-Gly-Phe-Arg-Gl
n-Ala-Ala-Ser-Val-Val-Val-Ala-Met-Asp-Lys-Leu-Arg-
Lys-Met-Leu-Val-Pro-Cys-Pro-Gln-Thr-Phe-Gln-Glu-As
n-Asp-Leu-Ser-Thr-Phe-Phe-Pro-Phe-Ile-Phe-Glu-Glu-
Glu-Pro-Ile-Phe-Phe-Asp-Thr-Trp-Asp-Asn-Glu-Ala-Ty
Examples thereof include those having a modified amino acid sequence that satisfies at least one of the conditions of r-Val-His-Asp.

The above-mentioned and the following description of amino acids and polypeptides in the present specification follow the abbreviations in the nomenclature or rules according to IUPAC and IUAC-IUB or the abbreviations commonly used in the art. The number or position of amino acids are all IL-1 even if there are deletions and additions.
It is displayed according to the amino acid sequence of β, that is, the sequence of the above formula (1). However, the numerical value indicating the position of an amino acid with a dash follows the amino acid sequence of formula (2).

Among the above IL-1β derivatives, preferred derivatives are those having an amino acid sequence satisfying at least one of the above requirements (a) to (c) and at least one requirement of (a) to (c) ( It has an amino acid sequence that simultaneously satisfies the requirement of d). The I
Specific preferred examples of the L-1β derivative are as follows.

At least position 1 ^Ala deleted or replaced with another amino acid residue, at least position 3 ^Val deleted or replaced with another amino acid residue, at least position 4 ^Arg deleted or replaced with another amino acid residue, at least 5 position ^Ser deleted or replaced with another amino acid residue, at least position 8 ^Cys deleted Substituted or substituted with other amino acid residues, at least ^{Arg at} position 11 deleted or substituted with other amino acid residues, at least ^{His at} position 30 deleted, or Substituted with another amino acid residue, at least position 71 ^Cys deleted or substituted with another amino acid residue, at least position 93 ^Lys deleted or another amino acid residue those substituted with a group, at least 97 of ^Lys deletion Which may or those substituted with other amino acid residues are those that at least 98 of ^Arg is substituted with one or other amino acid residues have been deleted, or both position 99 ^Phe is deleted less Substituted with another amino acid residue, at least 103- ^Lys deleted or substituted with another amino-acid residue, at least 120- ^Trp deleted or another amino acid residue Group substituted with at least 121 position ^Tyr deleted or substituted with another amino acid residue, at least position 153 ^Ser deleted or replaced with another amino acid residue , 1-position ^{Ala to} 3-position ^Val amino acid sequence, 1-position ^{Ala to} 6-position ^Leu amino acid sequence or 1-position ^{Ala to} 9-position ^Thr amino acid sequence at least deleted, 151-position ^{Val to} 153-position ^Ser amino acid sequence 149th
^Gln- 153 ^Ser amino acid sequence, 145 ^Asp- 153
Position ^Ser amino acid sequence, 141 position ^{Gln to} 153 position ^Ser amino acid sequence, 121 position ^{Tyr to} 153 ^Ser amino acid sequence or 1
^Lys No. 03 to ^Ser No. 153, at least the amino acid sequence deleted, those having a few amino acid residues at the N-terminus of formula (1), the amino acid sequence of formula (2) at the N-terminus of formula (1) Of 11
2 'position ^{Ala ~116'} position ^Asp amino acid sequence, 77 'position ^Met
-116 'position ^Asp amino acid sequence, 71' position ^{Met ~116} '
Position ^Asp amino acid sequence, 32 'position ^Met -116' position ^Ser amino acid sequence or 1 'position ^Met -116' position which at least has the ^Asp amino acid sequence.

The other amino acid residue in the above-mentioned IL-1β derivative capable of substituting and adding an amino acid residue at a specific position of the amino acid sequence of IL-1β may be any α-amino acid residue constituting a human body protein. Any neutral amino acid residue may be used, and the neutral amino acid residue is preferable. However, ^Cys may form an intramolecular or intermolecular disulphide bond based on its SH group, and in consideration of this, the amino acid residue is preferably the above amino acid residue other than ^Cys . Particularly preferred is ^{Arg at} position 4, for example.
If ^{Gly, Lys, Gln,} or ^Asp, the case of the 8-position ^{Cys Ser} or of
^Ala , ^Gln for 11th ^Arg , and ^His for 30th ^His
Tyr is 71 position ^Cys , ^Ser , ^Ala or ^Val is 93 position ^Lys
Leu or ^Asp in case of 98, ^Leu in case of ^Arg , 103 in case of ^Arg
Gnl for ^Lys , 120 ^Arg for ^Trp , 121st for ^Trp
Gln for ^Tyr , and ^{Met for} N-terminal addition,
Examples include ^Leu , ^{Arg, and Asp} .

Among the above IL-1β derivatives, at least the ^Cys at position 71 has been substituted or deleted, in particular the above ^Cys at another amino acid residue,
For example, those substituted with ^Ser , ^Ala , ^{Val and the} like show high activity. The at least 4-position ^Arg, 93-position ^Lys, 8-position ^Cys substitution or that what was deleted and at least 103 after position of at least one amino acid residue deleted are all such heating effects It has the characteristic of few side effects and toxicity. Furthermore, the compound of the formula (1) to which at least a specific amino acid residue or polypeptide is added to the N-terminal has low toxicity and is more effective as a drug in terms of duration of action.

The above specific IL-1β derivative can be produced by a genetic engineering technique. That is, it can be produced by utilizing a gene encoding the above-mentioned specific polypeptide, incorporating the gene into a microbial vector, and replicating, transcribing, and translating in the microbial cell.
This method is particularly advantageous in that it can be mass-produced.

The gene used in the above can be totally synthesized by chemical synthesis of nucleic acid according to a conventional method such as the phosphite triester method [Nature, 310 , 105 (1984)].
It is easy to synthesize using a gene encoding β or its precursor, and easily produced by, for example, modifying the nucleic acid sequence encoding the specific amino acid sequence from the gene according to a conventional method including the chemical synthesis means. it can. Incidentally, I
The gene encoding L-1β or its precursor is known (Japanese Patent Laid-Open No. 62-174022).

The above-mentioned nucleic acid (base) sequence modification operation can also be performed according to known methods,
It is carried out according to the amino acid sequence of the desired polypeptide [as a genetic engineering technique, for example, Molecular
Reference is made to Cloning Cold Spring Harbor Laboratory (1982). ] For example, conventional means such as restriction enzymes for the purpose of cutting, binding, phosphorylation of DNA, treatment of various enzymes such as DNA ligase, polynucleotide kinase, DNA polymerase and the like can be adopted. Available at. Isolation and purification of the gene or nucleic acid in each operation can also be performed by a conventional method such as agarose electrophoresis. Replication of the obtained gene can be performed according to a method using an ordinary vector. A DNA fragment encoding a desired amino acid sequence and a synthetic linker can be easily produced by the above-mentioned chemical synthesis. In the above, the codon corresponding to the desired amino acid is known and its selection may be arbitrary, for example, it may be carried out according to a conventional method considering the codon usage frequency of the host to be used [Nucl.Acids.Res., 9 , 43. -74 (1981)]. The partial modification of the codon of the nucleic acid sequence is, for example, 15 to 3 as usual.
Site-Specific Mutagenesisis (Site-Specific Mutagenesi) using a primer consisting of a synthetic oligonucleotide encoding a desired modification of about 0 mer
s) [Proc. Natl. Acad. Sci., 81 , 5662-5666 (1984)] and the like can be adopted.

The desired gene obtained by the above is, for example, the chemical modification method of Maxam-Gilbert [Maxam-Gilbert, Meth. Enzym., 65 , 4
99-560 (1980)] or M13 Phage dideoxynucleotide chain termination method [Messing, J. and Vieira, J., Gene, 1
9 , 269-276 (1982)] and the like to determine and confirm the nucleotide sequence. Thus, a gene encoding a polypeptide having the above-mentioned specific amino acid sequence is provided (hereinafter referred to as "target gene").

The IL-1β derivative utilizes the above-mentioned target gene and is a recombinant DN capable of expressing the target gene in a host cell.
It can be produced by preparing A, introducing it into a host cell for transformation, and culturing the transformant. Here, as a host cell, either a eukaryote or a prokaryote can be used. The eukaryotic cells include cells such as vertebrates and yeasts, and examples of vertebrate cells include ^Cos cells that are monkey cells [Y. Gluzman, Cell, 23 , 175-182 (1981)]. And Chinese hamster ovary cell dihydrofolate reductase-deficient strain [G. Urlaub and LA Chasin, Proc. Natl. Ac
ad.Sci., USA, 77 , 4216-4220 (1980)] and the like are often used, but not limited thereto. As an expression vector for vertebrate cells, a vector having a promoter located upstream of a gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc. can be used. May be owned. An example of the expression vector is pSV2dhfr [S. Subramani, R. Mulligan a which carries the SV40 early promoter.
nd P. Berg, Mol. Cell. Biol., 1 (9), 854-864 (1982)] and the like, but not limited thereto.

Yeast is generally used as a eukaryotic microorganism, and among them, yeast of the genus Saccharomyces can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as yeast include pAM82 [A. Miyan which has a promoter for the acid phosphatase gene.
ohara et al., Proc.Natl.Acad.Sci., USA, 80 , 1-5 (19
83)] and the like can be preferably used.

Escherichia coli or Bacillus subtilis is generally used as a prokaryotic host. For example, a plasmid vector capable of replicating in the host bacterium is used, and a promoter and SD are provided upstream of the gene so that the target gene can be expressed in this vector. It is possible to use a discovery plasmid having a (Shine and Dalgarno) nucleotide sequence and an ATG necessary for initiation of protein synthesis.
As Escherichia coli as the above-mentioned host bacterium, Escherichia coli K12 strain and the like are often used, and as the vector, pBR322 is generally used, but it is not limited thereto, and various known strains and vectors can be used. Available. The promoter, for example Trypto Juan promoter, P _L promoter, lac promoter, can be used lpp promoter, capable of expressing a gene of interest in each case. Taking the case of using the tryptophan promoter as an example, the vector pTM1 having the tryptophan promoter and the SD sequence as an expression vector [Now text, Man, Metabolism, vol.22,289 (1
985)] using the restriction enzyme Cl existing downstream of the SD sequence.
A gene encoding a desired polypeptide, to which ATG is added, may be linked to the aI site, if necessary.

Not only the direct expression system but also a fusion protein expression system utilizing, for example, β-galactosidase, β-lactamase or the like can be used.

The expression vector thus obtained is introduced into a host cell and transformed therewith by a generally adopted method, for example, cells in a logarithmic growth phase are mainly collected and Ca
It is possible to adopt a method in which the vector is incorporated by treating with Cl ₂ so that the DNA is easily incorporated naturally. In the above, as is generally known, MgCl ₂ or RbCl may be further allowed to coexist in the medium in order to further improve the efficiency of transformation, and the host cells are transformed into spheroplasts or protoplasts before transformation. The method can also be adopted.

The desired transformant thus obtained can be cultured according to a conventional method, and the desired polypeptide is produced and accumulated by the culture. As the medium used for the culture, various media commonly used for ordinary cell culture, for example, L medium, E medium, M
9 medium and the like, and any of the media generally known such as various carbon sources, nitrogen sources, inorganic salts, vitamins and the like may be used. When the above-mentioned tryptophan promoter is used, it is preferable to use, for example, M9 minimal medium to which casamino acid is added so that the promoter works. For this medium, tryptic acid such as indoleacrylic acid is used at an appropriate time. A drug for enhancing the function of the tophan promoter can also be added.

Purification and isolation of the desired polypeptide from the thus obtained culture containing the IL-1β derivative is carried out by various treatment operations utilizing the physical and chemical properties of the polypeptide [for example, "Biochemical Data Book II" pp1175. -1259, 1st edition, 1st printing, June 23, 1980, published by Tokyo Kagaku Doujin Co., Ltd.]. In extracting the polypeptide from the host, it is preferable to use mild conditions such as osmotic shock method from the viewpoint of maintaining the higher order structure. Specific examples of the above-mentioned purification and isolation methods include treatment with usual protein precipitants, ultrafiltration, molecular sieve chromatography (gel filtration), liquid chromatography, centrifugation, electrophoresis, and affinity detection. A combination of romatographies, dialysis methods, etc. can be adopted. More specifically, the above operation can be performed as follows, for example.
That is, first, the target polypeptide is partially purified in advance from the culture supernatant. This partial purification is carried out, for example, with an organic solvent such as acetone, methanol, ethanol, propanol, dimethylformamide (DMF), acetic acid, perchloric acid (PCA),
Treatment using an acid such as trichloroacetic acid (TCA) as a protein precipitation agent, treatment using a salting-out agent such as ammonium sulfate, sodium sulfate, sodium phosphate and / or a dialysis membrane,
It can be carried out by ultra-through treatment using a flat plate membrane, a hollow fiber membrane or the like. The operations and conditions of each process may be the same as those of ordinary processes. Then, 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 gelling agent used here is not particularly limited, and for example, any one made of dextran gel, polyacrylamide gel, agarose gel, polyacrylamide-agarose gel, cellulose or the like can be used.

The target polypeptide is obtained by subjecting the active fraction obtained by the above gel filtration to affinity chromatography using a hydrosiapatite column, DEAE method,
It can be further purified by subjecting it to ion exchange column chromatography such as CM method or SP method, chromatofocusing method, reversed-phase high performance liquid chromatography, or the like, or can be further purified by a combination of respective operations and isolated as a homogeneous substance. .

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

The pharmaceutical preparation of the present invention, that is, the recovery accelerator for persistent adrenal insufficiency subsequent to the steroid hormone excess state, has the above human IL-1β or its derivative as an active ingredient, and the same as the usual pharmaceuticals containing this kind of physiologically active substance. Can be one. That is, the pharmacologically effective amount of the above-mentioned active ingredient is usually
It is prepared into a practical drug form by using together with conventional ingredients in the formulation, for example, fillers, fillers, binders, wetting agents, excipients or diluents such as disintegrants, and other additives. Examples of the other additives used here include human IL
Human serum albumin (HS
A) and the like, L-type amino acids such as glycine and the like, and sugars (such as monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, sucrose, maltose and lactose) Disaccharides, dextran, polysaccharides such as hydroxypropyl starch, etc.), and conventional sulfur-containing reducing agents (eg, cysteine, N-acetylhomocysteine, thioctic acid, thioglycol) that can further increase the stabilization of the IL-1β active substance. Acids and salts, thioethanolamine,
Thioglycerol, thorium thiosulfate, thiolactic acid, dithiothreitol, glutathione, etc., and ionic and nonionic surfactants (for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type) , Fatty acid glycerides, etc.), and these may be used alone or in combination of two or more. The diluent is usually distilled water for injection, which may be various buffers such as citric acid-sodium phosphate, citric acid-sodium citrate, acetic acid-sodium acetate, disodium phosphate-.
PH of monosodium phosphate, citric acid-boric acid, etc.
8 may be used, and by using such a buffer solution, a stable isotonic preparation can be obtained.

The form of the above pharmaceutical composition is IL-
There is no particular limitation as long as it effectively contains 1β or its derivative, and it may be a solid preparation such as tablets, powders, granules and pills, and injections such as liquid preparations, suspension preparations and emulsion preparations. It may be in the form. It can also be made into a dry product which can be made into a liquid form by adding an appropriate carrier before use, and any of these formulations can be prepared by a conventional method.

The agent of the present invention containing human IL-1β or a derivative thereof thus obtained as an active ingredient has a suitable administration route according to its formulation form, for example, intravenous in the case of injection form,
It is administered by intramuscular, subcutaneous, intradermal, intraperitoneal administration, etc., and the pharmaceutical preparation in solid form can be administered orally or enterally. The amount of the active ingredient in the preparation and the dose of the preparation are appropriately selected depending on the administration method, the dosage form, the purpose of use, the patient to which the preparation is applied, and the like, but the amount of the active ingredient is usually about 0.
It is desirable to prepare a preparation form containing about 0,0001 to 80% by weight, and to administer this preparation in an amount such that the amount of the active ingredient contained therein is about 0.001 µg to 100 µg per adult per day. The administration need not be once a day and can be divided into 3 to 4 times.

Incidentally, by administration of the agent of the present invention, as an exogenous steroid hormone that causes secondary adrenal insufficiency or failure and adrenal atrophy capable of measuring healing or recovery, common steroid agents, namely cortisone, hydrocortisone (cortisol) Others include prednisone, prednisolone, methylprednisolone, triamcinolone, parametazone acetate, dexamethasone, betamethasone, etc.According to the present invention, secondary adrenal insufficiency or insufficiency and adrenal atrophy caused by administration of various steroid agents according to the present invention, It can be healed or recovered in a short period of time.

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

Example 1 Preparation of Secondary Adrenal Function Decrease Recovery Promoter IL-1β [1-148] (IL-1β Formula (1)
Of the amino acid sequence of the amino acid sequence of ^Gln- 153 of ^Ser.
No. 8), physiologically active saline (as GIF activity 1
× 10 ⁶ units / m), human serum albumin (HS
A) was added to 0.5% and the excess (0.22
Aseptically use 1 μm membrane filter)
Each m was dispensed into a vial and freeze-dried to prepare a preparation for injection.

The preparation thus obtained is 1 m distilled water for injection just before use.
It is used by dissolving in.

Example 2 Recovery-accelerating effect test of IL-1β derivative on secondary adrenal atrophy A group of 8 5-week-old SD male rats was used.

As a steroid administration group, 484.9 mg / of corticosterone was mixed in drinking water and administered for 2 weeks (dose: about 9.6 mg / day / rat).

As a control group, the steroid non-administration group was set up. Rats in this group were administered only drinking water for the same period.

Two weeks later, 0.2 m of physiological saline containing 0.1% bovine serum albumin (BSA) was added to the non-administered group, and this was further divided into two for the steroid-administered group. For IL-1β [1-148], 0.2 m of the above-mentioned BSA-containing physiological saline was further divided into two groups for the steroid drug administration group, and one of them was IL-1β [1- 14
8] at 1 μg per 0.2 m of the BSA-containing physiological saline solution
The test reagents prepared in the above amount were intraperitoneally administered twice a day in the morning and in the afternoon (experimental group). IL for the other one
The same amount of only BSA-containing physiological saline solution containing no -1β derivative was intraperitoneally administered in the same manner (comparative group).

The rats in each group were sacrificed on the 2nd, 4th and 7th days from the end of the administration of the steroid (the 0th day),
The adrenal gland was removed, its weight (mg) was measured, and the body weight was measured.

The results of adrenal gland weight obtained for each group of rats are shown in Table 1 below.

The adrenal gland weight is expressed as the mean value ± SE (n = 8) of rats in each group, and the * 1 mark in the experimental group's displayed value is p <0.05 compared to the comparison group, * 2 is the comparison. P <0.
Indicates 001. The dynamic * 1 mark is p <0.
05, * 2 indicates p <0.01 with respect to the control group.

In addition, as a result of the body weight measurement, administration of the steroid drug for 2 weeks showed a weight loss of about 12% as compared with the non-administration group, but during the observation period of 7 days after the administration, the steroid administration group Both the experimental group and the comparative group showed about 82 to 88% of the body weight of the steroid-non-administered group (control group).

Example 3 Fever Inducing Test of IL-1β Derivative GIF activity of human IL-1β derivative (50% maximum U / m
g) and LAF activity (50% max U / mg) as well as the exothermic induction of each of these derivatives was investigated as follows.

The GIF activity was measured according to the method described in the above-mentioned patent publication, and the LAF activity was measured by the method of Oppenheim [JJOppe
nheim et al., J. Immunol., 116 , 1466 (1976)], C
L measured using thymocytes of 3H / HeJ mouse
It was indicated by AF activity. The fever-inducing effect was obtained by subcutaneously administering to SD rats (male, 160 to 220 g) a solution prepared by dissolving a predetermined amount of each derivative in PBS (-) containing rat serum albumin at a ratio of 200 μg / m, and chronologically. Further, the body temperature of each rat was measured, and 6 hours after the rise in the body temperature was highest, the fever-inducing force was evaluated according to the following criteria.

<Evaluation of heat-induced force> -... 0 ≤ Δ ° C <1 + ... 1 ≤ Δ ° C <2 ++ ... 2 ≤ Δ ° C <3 The obtained results are shown in Table 2.

From the above table, it can be seen that all the tested human IL-1β derivatives have extremely reduced heat-inducing action and are excellent in safety.

Claims (2)

[Claims] 1. A preventive and recovery-promoting agent for secondary adrenal insufficiency, which comprises at least one selected from human interleukin 1β and its derivatives. 2. A human interleukin 1β derivative is human I.
The agent for preventing secondary adrenal insufficiency and promoting recovery as claimed in claim 1, which is L1β [1-148].