JPH08176008A - Neurotrophic agent - Google Patents

Abstract

PURPOSE: To obtain a neurotrophic agent, containing an interleukin 11 which is a simple protein comprising 178 amino acid residues as an active ingredient and useful as a therapeutic agent for neuropathy or a reagent for research of a tissue in the nervous system. CONSTITUTION: This new neurotrophic agent contains an interleukin 11 produced from a natural cell or prepared by using a gene recombination technique as an active ingredient. The agent is effectively used as a medicine or a reagent for providing a trophic factor for neurons. The action concentration range of the interleukin 11 used as the active ingredient is preferably 0.01-1μg/ml. This neurotrophic agent is useful for diagnosing or treating senile neuropathy represented by senile dementia.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel neurotrophic drug which serves as a trophic factor for nerve cells as a drug or a reagent.

[0002]

2. Description of the Related Art Motor neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehring disease), peripheral neuropathy due to diabetes and aging, or neurological diseases such as damage to the cerebrospinal and peripheral nerves It often follows a chronic course and belongs to the category of intractable diseases. In particular, as the population ages, the diagnosis and treatment of senile neurological diseases represented by senile dementia are becoming a major social problem to be solved.

Since most neurological diseases are thought to be caused by functional decline of nerve cells, so-called brain metabolism activating agents that improve cerebral vascular circulation or improve oxygen consumption and glucose metabolism of brain cells are therapeutic agents. Used.

On the other hand, until recently, it has been revealed that some cytokines have a neurotrophic effect, and they include nerve growth factor (NGF), brian-derived neurotophic factor, BD
NF), fibroblast growth facto
r, FGF), neurotrophin 3, N
T3) etc. have been reported (for example, a review by Kiyofumi Asai and Taiji Kato: Proteins, nucleic acids, enzymes, 36 , 1220-1226,
1991).

Interleukin 3 (IL-3) (Kamegai, M., et a, discovered as a hematopoietic cytokine)
l., Neuron, 4 , 4,29-436,1990) and interleukin 6
(IL-6) (Hama, T., et al., Neurosci. Lett., 1
04 340-344,1989), granulocyte / macropharge-colony stimulat
ing factor, GM-CSF) (Kamegai, M., et al., Brian Re
s., 532 , 323-325, 1990) was also recently found to have activity as a neurotrophic factor.

[0006] Each of these cytocans acting on the nervous system is a polypeptide and has an action mechanism different from that of a so-called low-molecular-weight cerebral metabolism activating agent because it expresses its action by binding to a specific receptor. There is a high possibility that they may be used alone or in combination with a low-molecular-weight metabolic activator as a novel therapeutic agent for neurological diseases.

[0007]

Some low-molecular weight brain metabolism activating agents for the treatment of cranial nerve diseases are being put to practical use (for example, idebenone, inderoxazine hydrochloride, bifemelane hydrochloride, aniracetam, pronto). (Fillin, etc.), its medicinal effect is not yet sufficient, and research is continuing to improve it.

[0008] Polypeptide neurotrophic factors represented by cytokines capable of acting on the nervous system are expected to be applied and developed in the future. On the other hand, efforts are being made to find new and useful neurotrophic factors. It continues.

[0009] As described above, trophic factors acting on the nervous system have not yet been fully elucidated, and the search for new and highly useful substances remains as a further problem.

[0010]

The present invention has been completed as a result of earnest research for solving the above problems. That is, the present invention is a neurotrophic drug containing interleukin 11 (hereinafter abbreviated as IL-11) as an active ingredient.

IL-11 is a simple protein consisting of 178 amino acid residues and is a bone marrow stromal cell line PU of the macaque monkey.
-34 was discovered as a growth stimulator of plasmacytoma cells (Paul, SR, et al., Proc. Natl. Acad. S
ci USA, 82 , 7512-7516, 1990), and an adipocyte differentiation inhibitory factor that suppresses the differentiation of preadipocytes into adipocytes (Kawashima, I., et al., FEBS lett. 283, 199-202, 1
991). Then, as a biological action of IL-11, in the presence of IL-3, a colony of megakaryoblast precursor cells (Bruno, E. et al., Exp. Hematol., 1
9 , 378-381, 1991) and bone marrow cell colonies (Musashi,
M., et al., Proc. Natl. Acad. Sci. USA, 8 8, 765-76
9, 1991) and the induction of acute phase proteins in hepatoma cell lines (Baumann, H and
Schendel, P., Biol. Chem., 266, 20424-20427, 199
1) etc. have been reported. However, no report has yet been made on the activity of IL-11 to promote the proliferation of nervous system cells, which was revealed by the present invention.

IL-11, which is used as an effective component of a neurotrophic drug, may be produced from natural cells, produced by gene recombination technology, or chemically synthesized. Good and not particularly limited.

Examples of IL-11 producing cells include normal cells such as stromal cells, fibroblasts, adipocytes and vascular endothelial cells. IL-11 in tumor cells
If a continuously producing strain of is obtained, it can be efficiently produced. When producing using genetic recombination technology,
CHO (Chinese Hamster Ovary) cells, mouse C
127 cells and the like can be used as host cells. Similarly, it is also possible to use insect cells such as silkworm and night moth, and microorganisms such as Escherichia coli, Bacillus subtilis, and yeast. When produced by normal cells or tumor cells, the culture supernatant,
In the case of genetically modified insect cells or adults, the culture supernatant or the parasite extract is used, and in the case of genetically modified microorganisms, the culture supernatant or the parasite extract is used as a raw material and separated by various chromatographies. be able to. Any chromatography can be used as long as it has affinity for IL-11. For example, a column made of silicon dioxide or calcium phosphate as a material, a column having a hydrophobic group as a ligand, a metal chelate column, an ion exchange column, and the like, and a molecular sieving column can also be mentioned as a usable carrier.

Specific examples of the dosage form for oral administration include tablets, pills, powders, capsules, granules, syrups, emulsions and suspensions. Such a dosage form is produced by a method known per se and contains a carrier or an excipient that is usually used in the field of formulation. Examples of dosage forms for parenteral administration include ointments, injections, poultices, inhalants, suppositories, transdermal inhalants and the like.

The concentration range of IL-11 used as an active ingredient is 1 ng / ml to 100 μg / ml,
It is preferably 0.01 to 1 μg / ml.

When glucocorticoid is used,
Either natural products (cortisol (hydrocortisone), cortisosterone, cortisone, etc.) or chemical compounds (dexamethasone, prednisolone, etc.),
The working concentration range is 10 ⁻⁹ to 10 ⁻³ M, preferably 10 ⁻⁷ to 10 ⁻⁴ M.

The effective dose and frequency of administration of the neurotrophic drug of the present invention will differ depending on the administration form, age and weight of the patient, and the nature or severity of the condition to be treated, but usually 0.
1-100 μg / kg, preferably 1-10 μg / kg
Can be administered once or in several divided doses.

The quantification of IL-11 according to the present invention is for inhibiting adipocyte differentiation (Ohsumi, J., et al., FEBS lett.
288 , 13-16, 1991) or growing mouse hybridoma 7TD1 cells in the presence of anti-IL-6 antibody (Fujie Shinkai et al., Biochemistry, Vol. 65, No. 8, P. 978, 1).
993) can be used as an index for quantification.

[0019]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Human normal diploid fibroblasts were transformed into "Cytodex 1" (Pharmacia, LKB Biotechnology, 3 g / 100 m).
After culturing at 37 ° C. on 1) and growing well, it was induced with synthetic nucleic acid poly I: poly C (10 μg / ml).

Using 12 liters of this culture supernatant (protein concentration: 292 μg / ml) as a purification raw material, a silica column "Microbeads silica gel", Fuji Devison, 30
φ × 200 mm). After washing with PBS (-),
300 ml of 20 mM hydrochloric acid (pH 2), then 500 ml
20 mM hydrochloric acid containing 25% ethylene glycol (pH
After washing with 2), containing 50% ethylene glycol 2
IL-11 was eluted with 100 ml of 0 mM hydrochloric acid (pH 2). The purity was 30% in a purity test by reverse phase HPLC using a C ₄ column (Vydac, 4.6φ × 250 mm).

Subsequently, this silica chromatography elution fraction was passed through an "S Sepharose column" (Pharmacia Biotechnology, 10φ × 20 mm), and then 60 ml of 20 mM hydrochloric acid (pH 2) and 20 mM phosphate buffer (pH 7). .4) 50 ml, 20 mM phosphate buffer (pH 8.0) 50 ml, 20 mM borate buffer (pH
9.0) 20 ml, 20 mM phosphate buffer (pH
8.0) After washing with 20 ml of 0.2 M sodium chloride 2
IL-1 in 20 ml of 0 mM phosphate buffer (pH 8.0)
1 was eluted. Purity was 60% in a purity test by reverse phase HPLC using a C ₄ column.

Further, the "S Sepharose column" elution fraction was adsorbed on a C ₄ column, and IL-11 was eluted with a gradient of acetonitrile concentration containing 0.1% trifluoroacetic acid. Purified IL-11 was eluted with about 60% acetonitrile. Overall yield by bioassay was about 40%. Laemmli's method for polyacrylic acid gel electrophoresis with sodium dodecyl sulfate (Nature,
227, 680-685, 1970), purified IL-11 showed a single band with a molecular weight of 24,000, and the purity by desymmetry was 95% or more. In addition, it showed a single peak on reversed-phase HPLC using a C ₄ column and had a purity of 96.
%Met.

Example 2 Measurement of proliferative activity of human neuroblastoma cells: Human neuroblastoma cell line NB-1 in a 25 cm ² plastic flask containing RPMI-1 containing 10% fetal calf serum (FCS).
The cells were cultured in 640 medium at 37 ° C. and 5% CO ₂ until they became confluent. NB became confluent
1 cell was detached by 0.25% trypsin treatment, 0.25%
The concentration was adjusted to 2 × 10 ⁴ cells / ml with FCS-containing serum-free medium “Cell Grosser H” (Meguro Laboratory / Sumitomo Pharmaceutical Co., Ltd.). This cell suspension was added to a 24-well plastic plate (Corning) at 0.5 ml / well, and 50 μl of the purified IL-11 preparation was added thereto. Then, the cells were cultured at 37 ° C. for 3 days, and the number of cells was counted with a call counter ZM. In this measurement, 3 wells were used for one measurement condition, the cell number was calculated as a ratio (%) to the control, and at the same time, the standard deviation was obtained.

The results are shown in FIG. 1 (IL-11 growth promoting action on NB-1 cells). As is clear from FIG. 1, NB-1 was observed to promote growth depending on the concentration of IL-11.

[0026]

INDUSTRIAL APPLICABILITY The neurotrophic drug of the present invention containing IL-11 as an active ingredient can be used as a novel neurotrophic drug for polypeptides, as a therapeutic drug for neurological diseases, or as a reagent for studying nervous system tissues.

[Brief description of drawings]

FIG. 1 shows the growth promoting action of interleukin 11 on NB-1 cells.

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Claims (3)

[Claims] 1. A neuronutrient containing interleukin 11 as an active ingredient. 2. The neurotrophic drug according to claim 1, which comprises natural interleukin 11 as an active ingredient. 3. The neurotrophic drug according to claim 1, which comprises genetically engineered interleukin 11 as an active ingredient.