JPH09157182A - New therapeutic agent for ulcerative colitis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent, comprising a mammalian interleukin-1 receptor or its specific fragment as an active ingredient, capable of manifesting excellent suppressing actions on shortening of an alimentary canal due to ulcerative colitis, having high safety and useful for preventing and treating the ulcerative colitis. SOLUTION: This new therapeutic agent for ulcerative colitis comprises (A) a mammalian interleukin-1 receptor (e.g. the one derived from a human) or (B) a fragment of the ingredient (A) including a physiologically active part (e.g. a protein which is a soluble fragment of a human I type interleukin-1 receptor comprising an amino acid sequence of the formula) as an active ingredient. Furthermore, the agent can be intravenously or intramuscularly be administered and prepared as, e.g. a powdery pharmaceutical preparation for injection. The agent is preferably administered in 1-15mg daily dose for an adult, expressed in terms of the amount of the exemplified protein in the ingredient (B).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a drug for preventing and treating ulcerative colitis.

[0002]

Ulcerative colitis is an idiopathic inflammatory bowel disease of unknown cause characterized by symptoms such as diarrhea, bloody bleeding, erosion of the lesion site and contraction of the intestinal tract. In addition, it is known that many cases of this disease become chronic, and an active phase in which clinical symptoms such as bloody stools are observed and a non-active phase in which clinical symptoms are apparently disappeared are repeated. This disease tends to increase year by year with the westernization of living environments such as eating habits in recent years. However, up to now, no sufficient treatment method has been established, and in the current situation, in the severe cases, it is unavoidable to resort to surgical therapy, and in Japan, the Ministry of Health and Welfare has designated the intractable disease. As conservative therapy,
In mild and moderate cases, salazosulfapyridine and steroids can be administered as oral agents, suppositories, or enemas, respectively. In severe cases, a large amount of steroid is intravenously administered under proper systemic management (Iwasaki Sasaki, Pharmaceutical Journal, vol.28, p37-4).
1, (1992)).

However, salazosulfapyridine has side effects such as nausea, headache, fever, rash, hemolytic anemia, exfoliation of the epidermis, granulocytopenia, fibrous alveolitis, headache, pancreatitis and male infertility (Allegayer). Et al, Gastr
ointestinal pharmacology,
vol. 21, p643-658, (1992)), and various serious side effects of steroids are known (Sadao Kashiwazaki et al., Comprehensive Clinical Practice, vol.43, p1725).
-1729, (1994)). The existing therapies are not sufficient as therapeutic agents for ulcerative colitis, and there is a need for therapeutic agents that are more effective than conventional therapeutic agents and that are highly safe.

[0004]

Therefore, an object of the present invention is to provide a protein which has an excellent effect on the treatment of ulcerative colitis and can be used as a highly safe new therapeutic drug. is there.

[0005]

The present invention relates to a therapeutic agent for ulcerative colitis, which comprises a mammalian interleukin 1 receptor or a fragment thereof including a physiologically active portion as an active ingredient. The present invention relates to a therapeutic agent for ulcerative colitis, in which a fragment of interleukin 1 receptor is a soluble fragment. The present invention relates to a therapeutic agent for ulcerative colitis, wherein the interleukin 1 receptor or a fragment thereof including a physiologically active portion is human interleukin 1 receptor or a fragment thereof. The present invention relates to a therapeutic agent for ulcerative colitis, in which the soluble fragment of human interleukin 1 receptor is a soluble fragment of human type I interleukin 1 receptor. The invention is human type I interleukin 1
Soluble fragments of the receptor are 1 to 3 of SEQ ID NO: 1 in Sequence Listing.
The present invention relates to a therapeutic agent for ulcerative colitis, which is a protein consisting of the 12th amino acid. The present invention also provides a mammalian interleukin 1 receptor or physiological agent for producing a therapeutic agent for ulcerative colitis, which comprises a mammalian interleukin 1 receptor or a fragment thereof including a physiologically active portion as an active ingredient. It relates to the use of fragments thereof which comprise the active part. The present invention further relates to a method of treating ulcerative colitis, which comprises administering to a patient with ulcerative colitis an effective amount of a mammalian interleukin 1 receptor or a fragment thereof that includes a physiologically active portion.

Interleukin 1 (hereinafter, interl
eukin-1: It may be abbreviated as IL-1. Shows various activities on a large number of cells and plays an important role in various reactions including the homeostasis of the living body in addition to the acute inflammatory reaction (Op).
penheim, J .; J. et al. , Immuno
l Today vol. 7, p. 45-56, 198
6).

1984, mouse IL-1 cDNA
Has been cloned (Lomedico, P. et al.
T. et al. , Nature, vol. 312,
p. 458-467, 1984), human, rabbit and rat IL-1 cDNAs were cloned and their structures were elucidated. IL-1 is a simple protein consisting of about 250 amino acids, the mature molecule has a molecular weight of 17.5 kD, and has no sugar. Unlike normal secretory proteins, IL-1 lacks a signal peptide, and therefore the mechanism of extracellular release has not been sufficiently clarified. It has been clarified that there are two molecular species of IL-1 in all animal species, and the alpha form (α
Type) and beta type (β type). IL-1 α
There is only about 25% homology in the amino acid sequence between IL-1β and IL-1β, but the same type I
Between L-1, there is a high homology of 60-70%. IL-1
α and IL-1β bind to the same receptor and show almost the same biological activity.

IL-1 comprises monocytes, macrophages and related cells (Langerhans cells etc.) of bacteria, endotoxin (LPS), complement-fixing immune complex, complement component (C).
3a, C5a), interferon (IFN), tumor necrosis factor (TNF), colony stimulating factor (CSF), transforming growth factor (transforming).
growth factor-b (TGF-b)),
It is produced by various stimuli such as viruses. In addition, keratinocytes (keratinocytes), NK cells,
T cells, B cells, vascular endothelial cells, mesangium (mes
angium cells, synovial cells, astroglia (as
troglia), neutrophils, fibroblasts and the like also produce IL-1. Therefore, a very large number of cells produce IL-1, and the IL-1 producing cells at the time of inflammation and at the time of non-inflammation are also different.

On the other hand, IL is used to exacerbate the pathological condition of ulcerative colitis.
-1 is believed to be deeply involved. It has been reported that the detection rate of IL-1 mRNA is significantly higher in biopsy mucosal tissues of patients with active ulcerative colitis than in non-active biopsy mucosal tissues (Isaacs et al. ，
Gastroenterology, vol. 103,
p1587-1595, (1992)). In addition, in the case of culturing macrophages obtained from a biopsy mucosal tissue of a patient with active stage ulcerative colitis, compared with the case of culturing macrophages obtained from a non-active period tissue, IL-
It has been clarified by a solid-phase enzyme-linked immunosorbent assay that the release amount of 1 is high, and it is shown that the IL-1 producing ability of macrophages of active stage ulcerative colitis patients is enhanced. (Mahida et al., Gut, vol.
30, p835-838, (1989)). That is,
Since IL-1 is closely involved in the inflammatory response in addition to the immune response, the enhancement of the IL-1 production ability in such patients with active stage ulcerative colitis increases the pathological condition of ulcerative colitis. It is considered to be deeply involved in the exacerbation, and it has been suggested that inhibition of the activity of overproduced IL-1 can treat ulcerative colitis (Dinarell).
o et al., TiPS, vol. 14, p155-159,
(1993)).

IL-1 activity inhibitors include IL-1 receptor antagonists (hereinafter sometimes abbreviated as IL-1RA), anti-IL-1 antibodies and IL-1 receptors (hereinafter IL-1R). May be abbreviated as.) And the like. Among them, regarding IL-1RA, Com
inelli et al.
y, vol. 103, p5-71, (1992)), T
homes et al. (Agents and action
s, vol. 34, p187-190, (199
1)), McCafferty et al. (Eicosanoi).
ds, vol. 5, p121-125, (1992))
And McCall et al. (Gastroenterology
y, vol. 106, p960-972, (199
4)) reported a therapeutic effect on an animal model of ulcerative colitis. On the other hand, anti-IL-1 antibody and IL-
Although it was hypothesized that 1R would effectively act against ulcerative colitis, which is considered to be caused by IL-1, by specifically inhibiting IL-1, it has never been confirmed in practice. Not (Sartor, Gast
roentology, vol. 106, p. 53
3-539, (1994)).

The IL-1RA, anti-IL-1 antibody and I
Of the three L-1Rs, IL-1RA inhibits the activity of IL-1 by competitively binding with IL-1R on the cell surface IL-1R in tissues in vivo. IL-1 antibody and IL-1R inhibit the activity of IL-1 by directly binding to IL-1 in tissue (Dinarello).
Et al., Blood Purif, vol. 11, p118
-127, (1993)). IL-1R on the cell surface is present in almost all organs in the living body (Dinar
ello, Blood, vol. 77, p1627-1
652 (1991)), IL-1RA may be trapped by other organs and may not reach the disease site. Furthermore, it is considered that this has an adverse effect on other organs. On the other hand, IL-
Cytokines such as 1 are characterized as acting locally, and this characteristic is considered to be a property greatly different from hormones (Junji Nishida, Molecular Hematology, p167).
-227, edited by Fumimaro Takaku, Nankodo, (1991). Therefore, it is considered that the site where the IL-1 producing ability is enhanced is limited to the disease site. Therefore, anti-IL-
1 antibody and IL-1R may act specifically on the disease site.
It is considered to be more useful than the above. Of these, anti-I
Regardless of its origin, L-1 antibody always has a problem of antigenicity upon administration, whereas IL-1R is originally present in the human body, and the problem of antigenicity is almost zero. It is considered not to exist.

[0012] Therefore, the inventors of the present invention considered that IL-1R has the most therapeutic effect on ulcerative colitis among IL-1 activity inhibitors, and as a result of diligent research, IL-1R and its cells were investigated. Soluble IL-1 which is a protein composed of external components
It was discovered that a fragment retaining at least physiological activity of R and IL-1R has a therapeutic effect on ulcerative colitis, and completed the present invention.

The IL-1R of the present invention is present in the body of mammals such as humans, monkeys and mice. Particularly preferred human IL-1R is 5 shown in SEQ ID NO: 1 in the sequence listing.
It consists of 69 amino acids. Preferred is a soluble fragment which retains its physiological activity. Soluble fragments include the extracellular portion of IL-1R. An example of a preferable soluble fragment is a protein consisting of amino acids 1 to 319 in SEQ ID NO: 1 in the sequence listing. More preferably, it is a soluble protein consisting of amino acids 1 to 312 (hereinafter abbreviated as shuIL-1R). This shuIL-1R is a known protein and is described in JP-A-5-339294. I
Two types of L-1 receptors are known, a type I having a molecular weight of about 80,000 and a type II having a molecular weight of about 68,000.
It is also known that it can bind to both α-type and β-type of active IL-1, and to both type I and type II receptors with different affinities. The type I IL-1R and its soluble fragment can be purified from mammalian leukocytes, but they are preferably produced by genetic engineering.
The genetic engineering manufacturing method is described in the above-mentioned patent publication. Since the IL-1R of the present invention is specifically produced by animal cells (Chinese hamster ovary cells: CHO cells), since it has a sugar chain as in vivo,
There are no side effects due to antigenicity, and the drug amount and administration method are easy to control.

The protein of the present invention is useful as a therapeutic agent for ulcerative colitis, and its administration method can be intravenous or intramuscular administration. In the case of intravenous administration, other intravenous infusion other than usual intravenous injection is used. Intravenous injection is possible. The therapeutic agent for ulcerative colitis of the present invention may contain a pharmaceutically acceptable carrier. The injection preparation may be, for example, an injection powder preparation. In that case, one or more appropriate water-soluble excipients such as mannitol, sucrose, lactose, maltose, glucose, fructose, etc. are added, dissolved in water, dispensed into vials or ampoules, and then lyophilized. It can be sealed to give a formulation. The clinical daily dose for an adult varies depending on the administration method, patient age, body weight, symptom, etc., but is usually 1 as shuIL-1R.
Is in the range of ~ 15 mg.

The therapeutic effect of the protein on ulcerative colitis is proved by examining the therapeutic effect of the protein on the dextran sulfate-induced mouse ulcerative colitis model. Animal models for ulcerative colitis include:
Known models include colitis model induced by injecting acetic acid or trinitrobenzene sulfonic acid into rat rectum, colitis model induced by intravenous administration of immune complex to rabbit, or dextran sulfate-induced mouse model used in the present invention. Kiyoro Kuroe et al., Medical History, vol.16
6, p63-66 (1993)). Among these, the dextran sulfate-induced mouse model used in the present invention is considered to be the most excellent animal model reflecting human pathological conditions (Okayasu et al., Gastroenterolo).
gy, vol. 98, p694-702 (199
2)).

In human chronic ulcerative colitis, it is known that the colon is shortened due to causes such as inflammation, edema, muscular hypertrophy, fibrotic tissue and lipid deposition (S).
umner et al., Immunological Dise
ase (second edition), Little
e, Brown and Company, Boston
n, vol. II, p1346-1366 (197)
1)). Shortening of the large intestine was also observed in an X-ray examination conducted in actual clinical practice, and it has been used as an important index for diagnosis (Shineko Tsuneo et al., Ulcerative colitis, Nankodo, p.
119-146 (1984)). In addition, one of the actions of leukotriene known to play an important role in the pathophysiology of ulcerative colitis is the contraction of smooth muscle, which results in shortening of the intestinal tract, It is thought that the diarrhea symptom is exacerbated by shortening the time taken for intestinal contents to transit through the intestine (Ciancio)
Et al., Ann. N. Y. Acad Sci. , Vol. 6
64, p210-221 (1992)).

The protein of the present invention exhibits species specificity, and it has been revealed that human soluble IL-1R does not act in rodents such as mice. Therefore, in the present invention, the inhibitory effect was examined by administering recombinant mouse soluble IL-1 receptor (type I) (hereinafter abbreviated as rmuIL-1R). From the results, this protein significantly suppressed the shortening of the length of the intestinal tract, which is one guideline for the effect of suppressing the development of ulcerative colitis. The dextran sulfate-induced mouse model used in the present invention is not limited to ulcerative colitis,
Even for Crohn's disease classified as the same inflammatory bowel disease, it is said to be an animal model that reflects the pathological condition (Kuroe Kiyoro et al., Medical Ami, vol.166, p63.
-66 (1993)). Therefore, from the results obtained this time,
The protein of the present invention is considered to be effective as a therapeutic agent for Crohn's disease. Therefore, the present invention relates to a therapeutic agent for Crohn's disease containing a mammalian interleukin 1 receptor or a fragment thereof including a physiologically active portion as an active ingredient. The present invention also relates to a method of treating Crohn's disease comprising administering to a patient with Crohn's disease an effective amount of a mammalian interleukin 1 receptor or a fragment thereof that includes a bioactive portion.

The effects of the present invention will be specifically described below with reference to examples.

【Example】

Example 1 Test using dextran sulfate-induced mouse ulcerative colitis model 9-week-old female CBA / J mice (purchased from Charles River Japan) 3% dextran sulfate (abbreviated as DSS, molecular weight: 54,000) The solution was purchased as drinking water freely for 10 days to prepare an ulcerative colitis model. 1 mg / kg / day and 0.2 mg / kg / d for 10 days from the start of DSS intake
The soluble IL-1R of ay mice was intravenously administered (hereinafter referred to as DSS + rmuIL-1 group). Note that rm
In accordance with the method described in Japanese Patent Laid-Open No. 5-339294, uIL-1R has a DNA sequence encoding the same protein (SEQ ID NO: 3 in the Sequence Listing of Japanese Patent Laid-Open No. 5-339294).
It was prepared by expressing in O cells. In addition,
Since a mouse pathological model was used, mouse IL-1R was used because of its species-specific relationship. When diluting rmuIL-1R to the required concentration, phosphate buffer (hereinafter PBS)
Is abbreviated). In addition, mice that were ingested with DSS and intravenously administered with PBS alone (hereinafter referred to as DSS + PBS group) were used as a disease onset control group, and DSS-free distilled water was freely ingested as drinking water, and only PBS was used daily. Mice administered intravenously (hereinafter referred to as distilled water + PBS group)
Was used as a disease-free control group. This experiment was conducted with 10 animals per group. Mice were sacrificed 10 days after the start of drinking DSS, the intestinal tract length of the large intestine was measured, and the inhibitory effect on the shortening of the intestinal tract caused by colitis was observed. The results are shown in Table 1. The ** mark in the table is the target DSS.
It shows that p <0.01 for the + PBS group.

[0020]

[Table 1]

Dunnett's multiple comparison test method (Dunnet)
In the evaluation by statistical treatment using te two-tailed method (two-sided test), in the disease-onset control group (DSS + PBS group), the intestinal tract was significantly shortened as compared with the disease-free group (distilled water + PBS group). Admitted. DSS + rmuIL− compared to DSS + PBS group
In the 1R group, significant suppression of this intestinal shortening was observed (P <0.01). That is, 1 of rmuIL-1R
At mg / kg / day and 0.2 mg / kg / day, a significant inhibitory effect on intestinal shortening associated with colitis was observed.

[0022]

EFFECTS OF THE INVENTION The present invention provides a novel therapeutic agent for ulcerative colitis. This enables an ulcerative colitis treatment method that is effective and has few side effects.

[0023]

[Sequence list]

Claims (5)

[Claims] 1. A therapeutic agent for ulcerative colitis, which comprises a mammalian interleukin 1 receptor or a fragment thereof including a physiologically active portion as an active ingredient. 2. The therapeutic agent for ulcerative colitis according to claim 1, wherein the fragment of the interleukin 1 receptor is a soluble fragment. 3. The interleukin 1 receptor or a fragment thereof containing a physiologically active portion is human interleukin 1
The therapeutic agent for ulcerative colitis according to claim 1 or 2, which is a receptor or a fragment thereof. 4. The therapeutic agent for ulcerative colitis according to claim 3, wherein the soluble fragment of human interleukin 1 receptor is a soluble fragment of human type I interleukin 1 receptor. 5. The therapeutic agent for ulcerative colitis according to claim 4, wherein the soluble fragment of human type I interleukin 1 receptor is a protein consisting of amino acids 1 to 312 of SEQ ID NO: 1 in Sequence Listing.