JPH05213767A - Therapeutic agent for cerebral nerve disease - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for cerebral nerve diseases containing an interleukin 2 protein as an active ingredient and capable of promoting growth, survive, distension of neurite by directly acting on central nervous cell. CONSTITUTION:The objective therapeutic agent contains an interleukin 2 protein derived from human as an active ingredient and can be prepared in the form of an injection, a nasal absorbing agent, a percutaneous absorption agent, an oral agent, etc. The daily dose of the agent is about 0.001-100mug/kg expressed in terms of an amount of interleukin 2 protein and the agent is administered in 1-6 portions. Since the agent contains the interleukin 2 protein directly acting on cerebral nerve cell and exhibiting nutritional effect, the agent is different from a symptomatic treating agent such as cerebral metabolism activator or nerve circulation improver which has hitherto been used and has actions not existing in a well-known nerve nutritional factor. The agent is useful as a substantial therapeutic agent for a cerebro-vascular accident, Alzheimer syndrome, Parkinsonian disease, nerve injury, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for cranial nerve disease containing an interleukin 2 protein (hereinafter abbreviated as IL-2) as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Currently, as a therapeutic agent for cranial nerve diseases such as cerebrovascular disorder and Alzheimer's dementia, Parkinson's disease, brain damage, etc., which are the major diseases of the middle-aged and elderly, cerebral metabolism activating agents such as calcium fopanthenate and idebenone, Nicardipine,
Cerebral circulation-improving drugs such as cinnarizine and cerebral function-improving drugs such as levodopa are frequently used, but there are many unclear points about the mechanism of action of these drugs, the clinical therapeutic effect is insufficient, and symptomatic The reality is that it is used therapeutically. On the other hand, ischemia, trauma, which is the fundamental etiology of cranial nerve diseases,
To treat the disease by directly preventing it or promoting regeneration of generalized or specific areas of functional deterioration, degeneration, and necrosis of nerve cells due to aging or etiology of unknown cause. For the purpose, many attempts have been made to search and discover substances called so-called neurotrophic factor (hereinafter abbreviated as NTF) from the living body, which are substances that promote the growth, survival and neurite outgrowth of nerve cells, and apply them as pharmaceuticals. It is being appreciated. As a typical example, nerve growth factor (hereinafter referred to as NGF)
Abbreviated. However, recent studies have shown that it has an effect only on a limited number of nerve cells in the brain (Proceedings).
Of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA)
88 , 961-965, 1991). In addition, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BD
NF), ciliary neurotrophic factor, neurotrophin-3
, Etc. have been shown to have the action as NTF, but the details are not clear, and the more effective NTF
It is the current situation that is eagerly awaited.

On the other hand, IL-2 was discovered by Morgan et al. In 1976 as a T cell survival / proliferation stimulating factor (Science 19 ).
3 , 1007-1008, 1976), and its primary structure was clarified by Taniguchi et al. In 1983 (Nature 302 , 305-310, 1983).
It is a glycoprotein and activates T cells and activates the defense mechanism of the living body. Therefore, renal cancer, malignant tumors such as malignant melanoma, bacterial and viral infections, immunodeficiency such as AIDS, autoimmune disease, It has been shown to be useful for medical immunotherapy for Hodgkin's disease and the like (Japanese Patent Publication No.
No. 279) It is clinically applied as an anticancer agent, but it is not known at all as a therapeutic agent for cranial nerve diseases.

As described above, IL-2 is widely considered to be a molecule which acts on the immune system, but little has been clarified about the action on other systems of the living body, particularly on the central nervous system. Benveniste et al.
In vitro, it was reported that IL-2 acts as a growth factor for oligodendrocytes forming the myelin sheath of nerve fibers in the central nervous system (Natur (Natur)).
e) 321 , 610-613, 1986), Haugen et al. reported to promote neurite outgrowth of peripheral sympathetic nerve cells (Journal of Neuroscience Research (J. Neurosci.
Res. ) 25 , 443-452, 1990). Also,
Lapchuck et al.
-2, and the presence of the IL-2 receptor were suggested (Neuroscience. 44 , 1,
73-184, 1991). The above reports can be seen. However, it was described that IL-2 directly acts on the central nerve cells that play the most important role in the cranial nervous system, and in particular that it has neurotrophic effects such as survival maintenance of nerve cells and neurite outgrowth. No reports are known.

[0005]

The present invention provides a therapeutic agent for cranial nerve disease containing as an active ingredient a new neurotrophic factor (hereinafter sometimes abbreviated as NTF) that can be an essential therapeutic agent for the above-mentioned cranial nerve disease. That is the subject of the present invention.

[0006]

Means for Solving the Problems The present inventor is in the process of exploratory research on neurotrophic factors for nerve cells in the brain, and IL-2 directly interacts with cerebral nerve cells which have no effect on NGF and bFGF. The present invention has been completed by discovering a fact that has never been known so far that it acts and promotes its survival, and further earnestly researched.

That is, the present invention relates to interleukin 2
A therapeutic agent for cranial nerve diseases, which contains a protein as an active ingredient. Further, it is preferable that the IL-2 is of human origin. Hereinafter, the present invention will be described in detail. IL-2 as an active ingredient of the therapeutic agent for cranial nerve diseases of the present invention may be any substance as long as it has an IL-2-like activity, that is, an action capable of maintaining T cells for passage. For example, natural human IL-2 produced in the animal body or animal cells (Japanese Patent Laid-Open No. 60-34).
No. 917), human IL-2 produced by gene recombination technology (Japanese Patent Publication No. 1-24279), or related substances thereof. The above-mentioned IL-2 and these related substances may or may not have a sugar chain. As the above-mentioned IL-2, specifically, human IL shown by Taniguchi et al., Which is produced by a genetic engineering technique
-2 primary structure (Nature 302 ,
305-310, 1983) and fragments thereof consisting of a partial amino acid sequence necessary for biological or immunological activity. As the above fragment, a fragment lacking a part of the N-terminal on the primary structure of IL-2 (EPC Publication 9153)
9) or a fragment lacking a part of the C-terminal (JP-A-60-126088). Furthermore, a part of the constituent amino acids of the above-mentioned polypeptide is deleted or substituted with another amino acid (Japanese Patent Laid-Open No. 59-9).
No. 3093). In addition, Met may be present at the N-terminus of the polypeptide (Japanese Patent Laid-Open No. 61-7879).
No. 9).

The above IL-2 is chemically modified with succinylated IL-2 (JP-A-1-500829) or a polyethylene glycol (hereinafter abbreviated as PEG) derivative (JP-A-60). No. 226821). It can also be obtained as a commercially available purified product from Zenzyme, etc., and these may be used. As the above-mentioned IL-2, human IL-2 or a substance having human IL-2 like activity is desirable.

Further, the preparation of the present invention contains IL as an active ingredient.
It suffices that it contains -2, and it can be produced by any known pharmaceutical production method. The IL-2
Is a suitable carrier commonly used as a drug, or
Medium such as sterilized water or saline, vegetable oil (eg sesame oil, olive oil), harmless organic solvent (eg ethanol)
Etc., and if necessary, excipients (eg, mannitol), colorants, emulsifiers (eg, cholesterol), suspending agents (eg, gum arabic), surfactants (eg, polyoxyethylene hydrogenated castor oil type) Surfactants, polyethylene glycol-based surfactants), solubilizers (eg sodium phosphate), stabilizers (eg sugar, sugar alcohols, albumin)
Alternatively, it is appropriately combined with a preservative (paraben) or the like to prepare a pharmaceutical preparation suitable for effective administration to a living body, a nasal absorption agent, a transdermal absorption agent, an oral preparation, etc., preferably an injection preparation. You can do it. For example, the formulation of the injection can be provided in the form of a freeze-dried product, a water solution for injection, a form enclosed in an osmotic pump, or the like.

Since the preparation of the present invention contains IL-2 which directly acts on the nerve cells of the brain and exerts a nutritional effect, it is a symptomatic therapeutic agent such as a cerebral metabolism activating drug and a cerebral circulation improving drug which have been used until now. In contrast to the underlying causes of various cranial nerve diseases, such as ischemia, trauma, aging, or unknown causes, general or specific areas of functional decline, degeneration, and necrosis of nerve cells are directly addressed. In addition, it can be used for treating diseases by preventing it or promoting regeneration. Examples of the cranial nerve disease include cerebrovascular disorder, Alzheimer's dementia, Parkinson's disease, and brain injury,
Not limited to this. IL-2 which is the active ingredient of the present invention is i
maintaining the survival of cerebral nerve cells in n vitro,
Since it has an action that is not found in known neurotrophic factors such as NGF and bFGF, it can be used more effectively as a therapeutic agent for cranial nerve diseases. The fact that the preparation of the present invention is effective even in a living body means that, for example, Krome (Krome)
r) et al., brain injury pathological model (Science 235 , 214-216, 198) .
You can prove it with 7).

IL-2 is administered to humans at 5 μg / kg / day
No serious side effects were observed even after 40 days from intraarterial injection (Journal of the Japanese Association for Cancer Treatment, 24 , 2015, 198).
9) In addition, there is a report that no death occurred even after a single intravenous administration of 6 mg / kg to cynomolgus monkeys (Japanese Patent Laid-Open No. 64-85920), which shows high safety. There is.

The formulation of the present invention contains, for example, about 0.001 to 100 μg / kg of IL-2 protein per day per day.
It can be administered by a method such as intraarterial injection, intravenous injection, intramuscular injection, subcutaneous injection, intracerebroventricular injection, intrathecal injection, etc., divided into 6 times. Preferably 0.1-5 μg /
The dose to be kg / day is divided into three times a day, and is administered once or continuously from the intrathecal cavity. Further, IL-2, which is an active ingredient of the present invention, is added to a liposome-forming substance (JP-A-60-243).
No. 022 or JP-A-64-85920)
After encapsulation in, it can be administered as liposomes. Alternatively, a catheter can be implanted in the ventricles or the medullary cavity and the drug can be directly administered from this. Alternatively, it can be continuously administered by enclosing it in an osmotic pump or the like and leaving it in the living body.

In addition, when hypertonic solution such as mannitol or urea is injected from the carotid artery, the permeability of the blood-brain barrier is transiently increased (Procedures of the National Academy of Sciences of the United States of America (Proc. . Nat
l. Acad. Sci. USA) 76 , 481-48
5, 1979), and substances such as alkyl glycerol have been reported to have an action of promoting the transfer of other drugs to the brain (Anjuwande Chemie (Angew. C).
hem. ) 23 , 257-328, 1984), and can also be administered using such techniques. In addition, it has been suggested that cationized albumin that is cationized by chemically modifying albumin may be taken into the brain by some mechanism (J. Clin. Invest) 70 ,
289-295, 1982), IL-2 can be chemically modified using such a method and then administered.

Further, the therapeutic agent of the present invention can be used in combination with other NTF as appropriate.

Further, for administration of the therapeutic agent of the present invention to a living body at the time of transplantation of nerve tissue or nerve cells, or for preservation of nerve tissue or nerve cells before transplantation, maintenance of vitality, etc. It can also be treated with the therapeutic agents of the invention.

The present invention will be specifically described below with reference to experimental examples.

(Experimental example) IL-2 survival-maintaining effect on cerebral cortical neurons The method of Yoshida et al. (Neuroscience Letters (Neu)
rosci. Lett. ) 66 , 181-186, 19
According to 86), 16 to 18-day-old embryonic rats (obtained from Shizuoka Experimental Animal Agricultural Cooperative Association), neurons were isolated from the cerebral cortex of the brain, and DMEM / F12 medium containing 1% fetal calf serum (Japan Biomaterials Center). (Gibco Co.) and seeded on a culture plate pre-coated with polylysine (Sigma) at a density of 5 × 10 ⁴ cells / cm ₂ and cultured at 37 ° C. under 5% CO ₂ . After 24 hours, IL-2 was added to the culture medium of nerve cells. Recombinant human IL-2 and recombinant mouse I
L-2 is rat IL-2 and 2.
5S mouse NGF was purchased from Collaborative Research, and recombinant human bFGF was purchased from Imusera. After 48 hours, cells were fixed with a 1% glutaraldehyde solution, and the number of cells was determined by a colorimetric method using crystal violet (Analytical Biochemistry. 182 , 16-).
19, 1989). The results are shown in Table 1. The values are shown as the average value ± standard error of the percentage (%) of the number of surviving cells with respect to the number of seeded cells.

Table 1 ───────────────────────────── Sample (dose) Neuronal cell viability (%) ───── ──────────────────────── No addition (control) 22 ± 1 Human IL-2 (100 ng / mL) 89 ± 2 Rat IL-2 (10 ng) / ML) 93 ± 4 mouse IL-2 (10 ng / mL) 95 ± 5 mouse NGF (100 ng / mL) 21 ± 1 human bFGF (100 ng / mL) 23 ± 2 ───────────── ─────────────────

As is clear from Table 1, NGF and bFGF known as neurotrophic factors have no effect, but human, rat and mouse IL-2 clearly maintain survival of nerve cells. did.

[0020]

EXAMPLES Examples of the preparation of the present invention are shown below, but the present invention is not limited thereto.

Example 1 Human IL-2 0.3 mg Purified gelatin 50.0 mg Sodium phosphate 34.8 mg Sodium chloride 81.8 mg Mannitol 25.0 mg The above components were dissolved in 10 mL of distilled water for injection and sterile filtered. Then, 1.0 mL each was dispensed into a sterile vial and freeze-dried to prepare a preparation for injection.

Example 2 Human IL-2 0.3 mg Albumin 20.0 mg Sodium Phosphate 34.8 mg Sodium Chloride 81.8 mg Mannitol 25.0 mg The above ingredients were dissolved in 10 mL of distilled water for injection and sterile filtered. Each 1.0 mL was dispensed into a sterile vial and freeze-dried to prepare a preparation for injection.

[0023]

EFFECTS OF THE INVENTION The therapeutic agent of the present invention directly acts on central nerve cells and promotes growth, survival, and neurite outgrowth, so that it is a cranial nerve disease such as cerebrovascular disorder, Alzheimer-type dementia, Parkinson's disease, and brain injury. Can be used as an essential therapeutic agent such as.

In addition, for the purpose of administering the therapeutic agent of the present invention to a living body at the time of transplantation of nerve tissue or nerve cells, or for preserving the nerve tissue or nerve cells before transplantation and maintaining vitality, etc. It can also be treated with the therapeutic agents of the invention.

IL-2 which is an active ingredient of the therapeutic agent of the present invention
Is ischemia, trauma, which is the fundamental etiology of the above-mentioned cranial nerve diseases,
It is possible to directly prevent the dysfunction, degeneration, and necrosis of nerve cells due to aging or the etiology of unknown cause, or to promote the regeneration. It is different from symptomatic therapeutic agents such as circulatory improvers and has an action which is not found in known neurotrophic factors such as NGF and bFGF, and thus can contribute to medical treatment as an essential therapeutic agent for cranial nerve diseases.

Front Page Continuation (72) Inventor Masato Shimojo 977-4 Kawashimada, Gotemba City, Shizuoka Prefecture Sofu Dormitory B-302

Claims (2)

[Claims] 1. A therapeutic agent for cranial nerve disease containing interleukin 2 protein as an active ingredient. 2. The therapeutic agent for cranial nerve diseases according to claim 1, wherein the interleukin 2 protein is of human origin.