JP2688733B2 - Agent for preventing and treating gastrointestinal mucosal disorders - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to M-CSF (macrophage
colony stimulating factor: a pharmaceutical use of macrophage colony stimulating factor, and particularly to a preventive and therapeutic agent for gastrointestinal mucosal disorders containing the M-CSF as an active ingredient.

[0002]

2. Description of the Related Art It is known that gastrointestinal mucosal cells have a large turnover and are significantly affected by various tissue damage factors. A typical example of the tissue damage factor is a side effect of a drug administered for therapeutic purposes. For example, the occurrence of gastrointestinal mucosal damage due to the administration of various anticancer agents and antibiotics has been well reported since ancient times. The same applies to anti-inflammatory agents such as indomethacin and aspirin,
It is known that various gastrointestinal symptoms occur as side effects.

With respect to the above-mentioned gastrointestinal mucosal disorders, various effective treatments have been studied along with the role of the tissue-damaging factor that causes the disorder, but at present, the cause of the tissue-damaging factor is as much as possible. Only efforts have been made to remove it. Recently, as an attempt to actively prevent and treat gastrointestinal mucosal disorders, a glutamine-containing infusion solution has been developed and its effect has been noted and studied (JJPEN, Vol.14,
No. 6, pp.933-940 (1992)).

On the other hand, M-CSF is known as a biological factor having a biological activity of acting on normal bone marrow cells to promote differentiation and proliferation of macrophages / monocytes, and for example, prevention and prevention of diseases and pathological conditions involving leukopenia. It has been reported to be useful as a therapeutic agent, an adjunct to bone marrow transplantation, a preventive and therapeutic agent for various infectious diseases, a therapeutic agent for certain cancers, etc.
-2391 gazette). Further, M-CSF has been clarified to have an anti-inflammatory action and an anti-allergic action, and its usefulness as an anti-inflammatory agent and an anti-allergic agent has also been reported (see International Publication WO 91/08754).

An object of the present invention is the previously unknown M-
The purpose of the present invention is to develop new medicinal uses of CSF, especially for the prevention and treatment of the above-mentioned gastrointestinal mucosal disorders.

[0006]

Means for Solving the Problems In the course of researching various methods for preventing and treating gastrointestinal mucosal disorders, the present inventors have
We have found the unexpected fact that M-CSF is effective against the above-mentioned gastrointestinal mucosal disorders, completely different from the previously reported pharmacological actions. The present invention has been completed based on this novel finding.

According to the present invention, there is provided a preventive and / or therapeutic agent for gastrointestinal mucosal disorders, which comprises M-CSF as an active ingredient.

Application of the preventive and / or therapeutic agent for gastrointestinal mucosal disorders of the present invention (hereinafter simply referred to as "medicine of the present invention") exerts a protective effect against mucosal disorders and a pharmacological effect such as improvement of mucous membrane and digestive tract functions. To be done.

The gastrointestinal mucosal disorders to which the drug of the present invention is applied include various gastrointestinal mucosal disorders caused by various tissue damage factors. Among them, the medicament of the present invention is preferably applied to intestinal mucosal disorders, and thereby, its preventive and therapeutic effects can be exhibited.

The above-mentioned tissue damage factor comprehensively includes various factors that induce damage to gastrointestinal mucosal cells. Examples thereof include the above-mentioned various drugs and obstacle substances such as ethanol, long-term total parenteral nutrition (TPN), postoperative catabolism, and oxidative stress. Among them, the disorder caused by TPN is characterized as intestinal atrophy, and the mucosal disorders to which the drug of the present invention can be applied naturally include such intestinal atrophy and intestinal dysfunction state. The tissue-damaging factors of the above and gastrointestinal mucosal disorders resulting therefrom are well known in the art.

MC as an active ingredient in the medicament of the present invention
SF has an intrinsic physiological activity of M-CSF, that is, an activity of acting on normal bone marrow cells to promote differentiation and proliferation of monocytes and macrophages (see, for example, Science, 236 , 1229 (1987)).
Various physiologically active substances having The M-CS
Of course, F is not limited to a natural form, and may be a recombinant type obtained by a genetic engineering method, or a derivative thereof having the same activity.

Therefore, in the present specification, "M-CSF"
The term is intended to be used in a broad sense to comprehensively include the above natural types, recombinant types and their derivatives. Specific examples of the M-CSF that can be used in the present invention include, for example, European Patent Publication Nos. 261592 and 328061.
And those of human origin disclosed in WO 91/06567 and the like. For the sake of convenience, each of the above M-CSF is based on the amino acid sequence of the precursor consisting of 554 amino acids described in the above publications, and its 3
It is shown according to the notation that the 3rd amino acid Glu is the 1st position. As the M-CSF used in the present invention, those of human origin described in each of the above publications are preferable, but among them, the M-CSF has an N-terminal in the region from Glu 1 to Glu 5 and from Glu 145. M-CSF having an amino acid primary sequence having a C terminus in the region of Pro 214, particularly Thr 153 to Pro 214, is particularly preferable.
M-CSF having an amino acid primary sequence from al or Ser at position 4 to Thr at position 153 is most suitable for application as the pharmaceutical agent of the present invention.

The medicament of the present invention is usually M- as an active ingredient.
Prepared into a pharmaceutical composition containing a pharmacologically effective amount of CSF together with a conventional pharmaceutically acceptable non-toxic carrier,
It is administered by various routes depending on the form. As the formulation form, a liquid form such as a solution, suspension, emulsion or the like is generally used and can be advantageously employed. They are generally administered orally, intravenously, subcutaneously, intracutaneously, intramuscularly and the like. Preparation into each of the above-mentioned pharmaceutical forms can be carried out by an ordinary method, and the amount of the active ingredient compounded in the obtained preparation is usually in the range of about 0.001 to 100 mg protein. is there.

The medicine of the present invention is of course not limited to the above liquid form,
It is also possible to prepare various other dosage forms suitable for oral and parenteral administration which are usually adopted. Further, the pharmaceutical preparation may be prepared into a dry product form which can be made into a liquid form by adding an appropriate water-soluble carrier before use.

The dose of the drug of the present invention depends on the desired pharmacological effect,
It is appropriately determined according to the age, sex, degree of disability, etc. of the patient and is not particularly limited, but the active ingredient is usually about 0.001 to 50 mg / kg as a protein amount for adults.
The dose is preferably about 1 / day, and the preparation can be administered once or in multiple doses per day, or can be continuously administered by intravenous drip infusion. In particular, the drug of the present invention is preferably used as an appropriate infusion agent or a time-of-use adjusting agent that can be diluted with a normal infusion solution for continuous administration by intravenous drip infusion.

If desired, other pharmacologically active ingredients may be added to the medicament of the present invention. For example, a glutamine ingredient such as alanylglutamine is effective as such a combination agent.

[0017]

EXAMPLES The present invention will be described in more detail below with reference to test examples.

Test Example 1 M-CS produced by using Escherichia coli as the M-CSF according to the method described in International Publication WO 91/06567.
F (activator consisting of a polypeptide having an amino acid sequence from Ser at position 4 to Thr at position 153) was used.

The effect of M-CSF on gastrointestinal disorders
Kido's method (JJPEN, Vol.14, No.6, pp.933-940 (199
2)], the following tests were carried out using methotrexate (MTX) -injured rats.

That is, an intravenous catheter was placed in a male Wistar rat (Std Wistar / ST, Japan SLC) having a body weight of about 200 g, and an infusion solution having the following composition containing MTX was administered for 7 days to a gastrointestinal disorder model (control group). ) (MT
X dose: 0.3 mg / kg / day).

<Infusion composition> GE-3 (pharmacology and treatment, 20 volumes, special edition 2,645-655 pages, 1992) 140 ml Amiparene (registered trademark, manufactured by Otsuka Pharmaceutical Co., Ltd.) 89.2 ml Otsuka MV Injection (Otsuka Pharmaceutical Co., Ltd.) 0.1 ml MTX (manufactured by Takeda Pharmaceutical Co., Ltd.) 0.3 ml Distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) Total amount of 270 ml As a test group, an infusion solution prepared by adding 100 or 250 μg of M-CSF to the above infusion solution Administration group (M-CSF infusion group,
(N = 6 animals for each concentration of M-CSF administered).
-For each of the CSF infusion group and the above-mentioned infusion administration group without M-CSF as a control (control infusion group, n = 6),
A histological examination was performed to evaluate the effect of administration of M-CSF.

The histological examination was carried out by hematoxylin-eosin (HE) staining of tissue sections prepared according to a usual method and immunostaining using an antibody against nuclear proliferating antigen (PCNA).

In the above HE staining method, the tissue section is deparaffinized, washed with running water, subjected to nuclear staining with hematoxylin staining solution, colored in running water, and then stained with eosin staining solution according to a conventional method. Alcohol dehydration, xylene penetration, and filling with marinol were performed.

Immunostaining using the above anti-PCNA is
It was carried out as follows. That is, first, the tissue section was deparaffinized, and then 0.3% H ₂ O ₂ methanol solution (methanol 15
0 ml of 30% H ₂ O ₂ (1.5 ml) (prepared for use) was immersed at room temperature for 30 minutes to inactivate the endogenous peroxidase, treated with alcohol and transferred to water.
I got used to S. Blocking reagent (10%
Normal bovine serum, Seikagaku) was added, and after soaking at room temperature for 30 minutes, excess water was removed and the anti-human PCNA monoclonal mouse antibody (DAKO) was used as the primary antibody without washing.
-PCNA, PC10 (manufactured by Dako) was added, and immersed at room temperature for 1 hour. As a negative control, a blocking reagent was used instead of the above primary antibody. Then PB
After washing with S, secondary antibody (biotin-labeled anti-mouse IgG +
IgA + IgM, Seikagaku Corporation) was added, and the mixture was immersed at room temperature for 30 minutes. After washing with PBS, DAB-H ₂ O ₂ solution (3,3′-diaminobenzidine-4HCl (Wako Pure Chemical Industries, Ltd.) 30 mg, distilled water 135 ml, 0.5 M Tris HC
l buffer (pH 7.6) 15 ml and 2.5% H ₂ O ₂
It was soaked in 0.3 ml of an aqueous solution which was mixed at the time of use) for about 5 to 10 minutes to develop a color. As the blocking reagent, secondary antibody, and standard reagent, Histofine staining kit constituent reagents (Seikagaku Corporation) were used.

The results of the above test are shown in FIGS.

From the respective figures, the following is clear. That is, as small intestinal epithelial cell damage of MTX, mucosal cell swelling and cytoplasmic vacuole formation, epithelial cell detachment, plasma leakage into the intestinal lumen, leukocyte infiltration into the submucosa, and finally the entire length of the intestinal tract It is known to cause severe exfoliating enteritis. Also in this test, when the infusion solution containing MTX was performed for 7 days (control group), severe exfoliative enteritis of the jejunum was observed (see FIGS. 1 and 4).

However, the infusion solution containing MTX contains MC
When 100 μg or 250 μg of SF was added for 7 days (M-CSF infusion group), jejunal villi and crypt structures were retained in all cases, and exfoliative enteritis due to MTX could be effectively treated. (See FIGS. 2, 3, 5 and 6). The effect of M-CSF on the jejunum was also observed in the duodenum and ileum.

In the control infusion group, almost no PCNA positive cells were observed, whereas in the M-CSF infusion group, PCNA positive cells were found in the crypt cells (see FIGS. 7 and 8).

Test Example 2 This test demonstrates that M-CSF has a protective effect on the small intestinal mucosa in a small intestinal mucosal lesion model prepared by administering methotrexate to rats.

The mechanism of M-CSF's protective action on the small intestinal mucosa is considered to be the proliferative action of M-CSF on small intestinal epithelial cells. That is, the small intestinal epithelial cells are derived from undifferentiated cells in the crypts, and after the crypts undergo proliferative division, they differentiate into functional intestinal superficial cells while moving toward the lumen. After that, it falls off from the villus in 3 to 4 days. Therefore, the maintenance of the small intestinal mucosa depends largely on the proliferation of crypt cells. In order to confirm that the protective effect on the small intestinal mucosa by M-CSF is due to the growth stimulating effect on the small intestinal crypt cells, IEC-6 cells, which are small intestinal epithelial cell lines derived from rat small intestinal crypt cells, were used for the following in vitro. The test was conducted.

(1) Method IEC-6 cells were purchased from ATCC, 5% fetal calf serum (FCS), 0.1 U / ml insulin, 0.1 mg.
Subculture in DMEM medium containing 1 / ml streptomycin and 100 U / ml penicillin G.
Cells at passage -25 were used.

In the cell growth experiment, 4 × 10 ⁴ cells per well were cultured in a 24-well culture plate in a low serum medium (FCS of the above culture medium was 0.1%) for 12 hours. The cells were detached with a trypsin / EDTA solution, the number of cells was counted with a hemocytometer, and the number of cells was defined as the number of cells on day 0. Further, various concentrations of the M-CSF, epidermal growth factor (EGF: mouse, receptor gray; Becton
Dickinson), insulin (from bovine pancreas; Sigma), basic fibroblast growth factor (bFGF: human; Oncogene Science) and interleukin 1β.
(IL-1β; Kikumoto et al., Biochem. Biophys. Res. Commu
n, 147 , 315-321 (1987)) were added and the number of cells on the second and fifth days was measured in the same manner. Furthermore, M-CSF
The same experiment was repeated by adding various neutralizing antibodies (monoclonal antibody ANOC573: 1.2 mg / ml described in JP-A-5-95794) at various concentrations.

(2) Results EGF showing the strongest proliferative effect was shown in FIG. 9 with the number of cells cultured in DMEM containing 0.1% FCS as a control.
The result of comparing the proliferative effect by (0.1 μg / ml) with that by a typical growth factor insulin (0.1 IU / ml) and that by M-CSF (1 μg / ml) is shown. The value of each result is the average value ± SD (n =
9).

From the figure, it is clear that M-CSF has about half the potency of the action of EGF and exhibits a proliferative action almost equivalent to that of insulin.

In addition to the above growth factors, M-CSF
The proliferative effect of bFGF and IL-1β released by macrophages along with the activation of macrophages by the was also examined. That is, in the same way as above,
EGF, insulin, M-CSF, bFGF and IL-
1β was added at various concentrations, and the cell number on the 5th day was measured. The optimal concentration of bFGF (0.01 μg
/ Ml), heparin having a stabilizing effect on bFGF was added, and the cell number was similarly measured for its proliferative effect. Results obtained (mean ± SD (n =
9)) is shown in FIG.

From FIG. 10, it can be seen that M-CSF exhibits a proliferative action of about half that of EGF, and the optimum concentration is 1 μg / ml.

From the figure, it was confirmed that M-CSF has a proliferative action equivalent to or higher than that of insulin and bFGF. This proliferative effect was not observed with IL-1β.

Further, the above-mentioned proliferative action of M-CSF was confirmed by an inhibition experiment using a neutralizing antibody. That is, in the same manner as described above, cells diluted with 0.1 μg / ml of M-CSF were added with antibodies diluted 10, 50, and 100 times as final concentrations, respectively, and the number of cells on the 5th day was adjusted in the same manner. I asked. 1 μg / ml E as a positive control
The number of cells obtained by adding GF was also determined. The results (mean ± SD (n = 9)) are shown in FIG.

From FIG. 11, M-CSF was found to be a neutralizing antibody (5
It is clear that the proliferative effect of the IEC-6 cells is completely inhibited in the coexistence of 0-fold dilution or more).

As described above, M-C observed in vivo
The intestinal protection effect of SF on the small intestinal mucosa was confirmed by in vitro tests.
-Demonstrating that CSF is directly acting on crypt cells of the small intestine and promoting their proliferation, and it was also confirmed that when EGF-added proliferation was used as a positive control, it always had a titer of about half. Was done. From the above, it was revealed that M-CSF promotes the proliferation of epithelial cells, which is effective for protection against damage to the gastrointestinal mucosa due to various causes.

[0041]

INDUSTRIAL APPLICABILITY According to the present invention, a novel prophylactic and therapeutic agent for gastrointestinal mucosal disorders is provided, and gastrointestinal mucosal disorders caused by various tissue damage factors can be effectively treated.

[Brief description of the drawings]

FIG. 1 is a micrograph (× 40) of a tissue section after HE staining in a control infusion group (a photograph replacing a drawing showing the morphology of an organism).

FIG. 2 is a micrograph (× 40) of a tissue section after HE staining in the M-CSF infusion group (100 μg / kg) (a photograph replacing a drawing showing the morphology of an organism).

FIG. 3 is a micrograph (× 40) of a tissue section after HE staining in the M-CSF infusion group (250 μg / kg) (a photograph replacing a drawing showing the morphology of an organism).

FIG. 4 is a micrograph (× 100) of a tissue section after HE staining in a control infusion group (a photograph replacing a drawing showing a morphology of an organism).

FIG. 5 is a micrograph (× 100) of a tissue section after HE staining in the M-CSF infusion group (100 μg / kg).
(Photograph replacing a drawing showing the morphology of living things).

FIG. 6 is a micrograph (× 100) of a tissue section after HE staining in the M-CSF infusion group (250 μg / kg).
(Photograph replacing a drawing showing the morphology of living things).

FIG. 7 is a micrograph (× 400) of a tissue section after PCNA staining in the control infusion group (a photograph replacing a drawing showing the morphology of an organism).

FIG. 8 is a micrograph (× 400) of a tissue section after PCNA staining in the M-CSF infusion group (250 μg / kg).
Times) (a photograph replacing a drawing showing the morphology of living things).

FIG. 9 shows the epidermal growth factor (EG
F), M-CSF and IEC of insulin respectively-
It is the graph which investigated the proliferation effect with respect to 6 cells.

FIG. 10 is a graph showing IEC-6 cell proliferation effects of EGF, basic fibroblast growth factor (bFGF), M-CSF, insulin, heparin, and IL-1β shown in Test Example 2 described later.

FIG. 11 is a graph showing that the neutralizing antibody inhibits the same effect of M-CSF by using the IEC-6 cell proliferation effect of EGF shown in Test Example 2 below as a control.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Jun Mukai 1-9 Marusu, Hiroshima-shi, Matsushimo-cho, Itano-gun, Tokushima Prefecture (72) Inventor Masayuki Takahashi 79-7 Nakanokoshi, Kizu-no, Otsu-machi, Naruto-shi, Tokushima (56) ) References Tokuhei Hei 2-502192 (JP, A) International Publication 91/8754 (WO, A)

Claims (6)

(57) [Claims] 1. A preventive and / or therapeutic agent for gastrointestinal mucosal disorders, which comprises a pharmacologically effective amount of M-CSF together with a pharmaceutically acceptable non-toxic carrier. 2. M-CSF has 1st Glu to 5th Glu
Having an N-terminal at either position and at position 145 to Glu to 21
The preventive and therapeutic agent for gastrointestinal mucosal disorders according to claim 1, which has an amino acid primary sequence having any of Pro at position 4 as the C terminus. 3. M-CSF has 3rd Val to 153rd T
The preventive and / or therapeutic agent for gastrointestinal mucosal disorders according to claim 1, which has an hr amino acid primary sequence. 4. M-CSF is from Ser 4 to Ser.
The preventive and / or therapeutic agent for gastrointestinal mucosal disorders according to claim 1, which has an hr amino acid primary sequence. 5. The preventive and therapeutic agent for gastrointestinal mucosal disorders according to claim 1, which is a gastrointestinal mucosa protective agent and a gastrointestinal function improving agent. 6. The preventive and therapeutic agent for gastrointestinal mucosal disorders according to claim 1, wherein the pharmacologically effective amount is 0.001 to 50 mg protein amount / kg / day dose.