JP2009102226A - Therapeutic agent for spinal cord injury - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug for recovery from a spinal cord injury recovery exhibiting simple and high effects, to provide a food, a medicine composition containing the same, and to provide a spinal cord injury recovery method. <P>SOLUTION: Provided is Withanoside IV and its peripheral compounds, which are oxygen-containing heterogeneous ring-having steroid glycosides, can be isolated from Ashwagandha (root of Withania somnifera Dunal) and can be synthesized by chemical synthesis means, and have high spinal cord injury-recovering and treating effects. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for spinal cord injury, and particularly relates to a therapeutic agent for spinal cord injury containing withosideside IV and its peripheral compounds, a food / pharmaceutical composition containing the same, and a method for recovering spinal cord injury using the same. .

  There are over 100,000 people with spinal cord injury in Japan, and the number is said to increase by 5,000 each year. Many causes of spinal cord injury include traffic accidents, sports accidents, falls / falls from high places, etc.When these injuries occur, external forces such as flexion, extension, rotation and compression act on the spine in combination, Spinal cord injury as well as ligament injury results. (Spine and spinal cord injury, [searched on January 20, 2006], Internet <http://www.rd.mmtr.or.jp/-sumihosp/sekisonbun.htm>). In addition to trauma, there are spinal cord injuries and disc herniation.

  If the spinal cord is damaged, depending on how it is damaged, the degree of damage, and the site of damage, limb movement, paralysis of the sensation, and even severe respiratory problems may occur. The higher the location of the injury site (Sengoku → Lumbar → Chest → Neck), the wider the range of paralysis, the more severe the injury. For example, if the chest is damaged, the lower body can be paralyzed, and if the neck is damaged, all limbs can be paralyzed.

  Treatment methods for spinal cord injury currently include surgical methods, drug administration methods, etc., but they are not completely established, and in the case of complete injury, functional recovery is quite difficult. For example, a currently used treatment for acute spinal cord injury is a short-term steroid mass administration method (Non-patent Document 1). However, this method is difficult because it requires treatment within 8 hours after injury. In addition, there are documents that deny the steroid mass administration method itself, and it can be said that it has not been completely established. In addition, other methods such as transplantation of ES cells or bone marrow cells are currently being developed (Non-patent Documents 2 and 3).

  On the other hand, various studies are also being conducted on therapeutic methods using drugs for neurological dysfunction diseases. For example, Patent Document 1 proposes a therapeutic agent containing a substance secreted from dendritic cells or a substance that induces, proliferates, or activates dendritic cells as a therapeutic agent for nerve damage such as spinal cord injury. .

  Patent Document 2, Patent Document 3, and Patent Document 4 propose oxazopyrroloquinolines, pyrroloquinolines, indolequinone derivatives and the like as nerve growth factor production promoters.

Kronval E. et al., Lakartiddingen, June 2005, 13-26; 102 (24-25): 1887-8, 1890. Leroy PH. Et al., Blood Rev. November 2005, 19 (6), p. 321-331 Dezawa M. et al., Curr Mol Med. , November 2005, 5 (7), p. 723-732 JP 2004-002412 A Japanese Patent Laid-Open No. 06-009396 Japanese Patent Laid-Open No. 06-21660 Japanese Patent Laid-Open No. 07-118152

  However, since the therapeutic agent and the therapeutic method shown in the above-mentioned Patent Document 1 use dendritic cells or a subset thereof, they are not convenient and are difficult to standardize. In addition, any of the other methods and therapeutic agents described above are not completely established yet, and their effects are insufficient. Accordingly, in view of the background of the increase in spinal cord injury patients, development of a therapeutic method and / or therapeutic agent that exhibits a simple and high effect on spinal cord injury is strongly desired.

  Therefore, the objective of this invention is providing the chemical | medical agent which recovers the spinal cord injury which shows the simple and high effect, and the recovery method of a spinal cord injury.

As a result of intensive studies on the above problems, the present inventors made a spinal cord injury mouse as a model, and performed behavioral evaluation and immunohistochemical staining. As a result, with Witanoside IV and its peripheral compounds, a remarkable spinal cord injury recovery action As a result, the present invention has been completed.
That is, according to the present invention, it is possible to solve the above-mentioned problems by providing withanoside IV having a specific structure and its peripheral compounds.

  Therefore, as an aspect of the present invention, there is provided a therapeutic agent for spinal cord injury comprising at least one compound represented by the following structural formula 1 as an active ingredient.

Structural formula 1
(In the formula, R ₁ and R ₂ are each independently a hydrogen atom, a hydroxyl group, an alkyl group, or an alkoxy group, and R ₃ is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a sugar residue. )

  Among the therapeutic agents for spinal cord injury of the above structural formula 1, particularly preferred is a therapeutic agent for spinal cord injury containing the vitanoside IV of the following structural formula 2 as an active ingredient.

Structural formula 2

  Another aspect of the present invention is a food / pharmaceutical composition containing the therapeutic agent for spinal cord injury as an active ingredient and having an action of recovering spinal cord injury.

  Furthermore, another aspect of the present invention is a method for recovering spinal cord injury, comprising administering an effective amount of the therapeutic agent for spinal cord injury or a food / pharmaceutical composition.

  According to the present invention, it is possible to provide a therapeutic agent for spinal cord injury or a food / pharmaceutical composition using a specific compound that is simple and highly effective.

  Moreover, an effective therapeutic method for the purpose of treating spinal cords of humans or the like can be easily provided by the food or pharmaceutical composition containing the drug or an effective amount thereof.

  Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.

  In order to solve the above-described problems as described above, the present invention provides a therapeutic agent for spinal cord injury comprising at least one compound of the following structural formula 1 as an active ingredient.

Structural formula 1

In Structural Formula 1, R ₁ and R ₂ are each independently preferably a hydrogen atom, a hydroxyl group, an alkyl group, or an alkoxy group, and R ₃ is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a sugar It is preferably a residue. R ₁ and R ₂ are particularly preferably each independently a hydrogen atom or a hydroxyl group. R ₃ is particularly preferably a sugar residue.

  Examples of the alkyl group as the substituent are not limited thereto, but preferably include a linear or branched alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, Examples include propyl group, isopropyl group, butyl group, pentyl group, hexyl group and the like. Examples of the alkoxy group include, but are not limited to, the present invention, preferably a linear or branched alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, Examples thereof include an isopropoxy group, a butoxy group, an isobutoxy group, a pentyloxy group, and a hexyloxy group.

  Examples of the sugar residue as the substituent are not limited to these examples, but a glycosyl group from a monosaccharide, a sugar derivative, an oligosaccharide composed of 2 to 7 sugars, an oligosaccharide derivative, or the like is preferable. With respect to the bonds, the present invention is not limited to these, but α bonds and β bonds may be used, and α bonds and β bonds may be mixed. Specific examples of sugar residues include, but are not limited to, the residues of monosaccharides such as glucose, galactose, mannose, fructose, fucose, and arabinose; glucuronic acid, N-acetylglucosamine, N-acetylgalactosamine Residues of sugar derivatives such as sialic acid; maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, lactose, gentiobiose, xylobiose, primebellose, melibiose, lutinose, vicyanose, laminaribiose Residues of oligosaccharides composed of 2 to 7 sugars such as cellobiose, cellotriose and panose, and the like, and particularly preferred are residues of oligosaccharides composed of glucose and glucose. The hydroxyl groups at the 2, 3, 4, and 6 positions may be modified with, for example, an acetyl group, a malonyl group, a phosphate group, or a methyl group.

  Of the compounds represented by Structural Formula 1, Vitanoside IV represented by Structural Formula 2 below is highly effective and can be particularly preferably used.

Structural formula 2

  Withanoside IV and its peripheral compounds used as a therapeutic agent for spinal cord injury of the present invention can be isolated from Ashwagandha (root of Whitania soniifera Dunal). For example, Chem. Phar. Bull. 50, p760-765 (2002). Moreover, what was synthesize | combined with the chemical synthesis method can also be used, and the method is not specifically limited.

It can also be obtained as a metabolite of withanoside. For example, one of the compounds encompassed by structural formula 1 (somone, R ₁ = H, R ₂ = OH, R ₃ = OH) is a metabolite of witanoside IV, for example, Heterocycles, Vol. 34. , P689-698 (1992). Specifically, it can be obtained by treating Witanoside IV with an enzyme such as naringinase at pH 5.25 and 37 ° C. for 3 days, and separating and purifying it by partition high performance liquid chromatography.

  The food composition of the present invention can be applied particularly to food for patients with spinal cord injury. Spinal cord injury recovery treatment can be easily performed by ingesting directly as a special-purpose food such as a food for specified health use or a food with a nutritional function containing the drug of the present invention.

  Specifically, when used as a food composition, various foods and drinks (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, liquid foods, food for the sick The drug of the present invention may be added to foods such as infant milk powder, foods such as lactating milk powder, and nutritional foods. The present active ingredient can be used as it is, or can be used according to a conventional method for ordinary food compositions such as mixing with other foods or food ingredients. Moreover, about the property, the state of the food / beverage products normally used, for example, any of solid (powder, granule, etc.), paste, liquid or suspension may be sufficient.

  Other ingredients are not particularly limited, but foods and drinks containing the drug of the present invention mainly include water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Can be used as an ingredient. Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. Examples include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used in combination of two or more, and synthetic products and / or foods containing a large amount thereof may be used.

The therapeutic agent for spinal cord injury of the present invention The dosage of the food / pharmaceutical composition comprising this as an active ingredient is appropriately set in consideration of various factors such as the route of administration and the age, weight and symptoms of animals to be administered including humans. can do. Although this invention is not limited to this, Preferably 0.01-100 micromol / kg / day is suitable as an active ingredient.
The therapeutic agent for spinal cord injury and the pharmaceutical composition of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, etc.).

  Examples of the administration form of the spinal cord injury therapeutic agent and pharmaceutical composition according to the present invention include oral administration using tablets, coated tablets, capsules, granules, powders, solutions, syrups, emulsions and the like. These various preparations are prepared according to conventional methods, including the excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, solubilizers, suspension agents, and coatings. It can be formulated using known adjuvants that can be generally used in the pharmaceutical formulation technical field such as pharmaceuticals.

  The amount of the spinal cord injury therapeutic agent of the present invention can be arbitrarily determined according to the purpose and application (pharmaceutical composition such as prophylactic agent and therapeutic agent). Although this invention is not limited to this, As the content, 0.001 to 100% (w / w) normally with respect to the whole quantity, Especially 0.1 to 100% is preferable.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by these.

[Production of spinal cord injury model mice]
The dorsal skin and muscle of a mouse (ddY, male, 6 weeks old) were dissected under ether anesthesia to expose the spine. Next, the vertebral arch at level 8-11 of the thoracic vertebra was excised with an electric drill. A weight (2.5 g) was dropped 3 times from 2 cm vertically above the exposed thoracic vertebra, and the spinal cord was damaged.
On the other hand, the sham group was treated to the laminectomy.
At this time, in the spinal cord injury model mouse, both hind limbs were paralyzed, and the hind limbs were unable to support their weight and move. In addition, it was confirmed that the Sham group maintained normal motor function.

[Drug administration]
The first drug administration was performed 1 hour after the spinal cord was damaged. Subsequently, continuous oral administration for 20 days was performed once a day from the next day. Withanoside IV was dissolved in distilled water and administered at a dose of 10 mol / kg / day. In addition, water was orally administered as a vehicle to the Sham group and the control group. The witanoside IV used here is an isolated product after methanol extraction from Ashwagandha, and its purity was confirmed by HPLC (detection: UV 220 nm).

[Behavioral evaluation]
The following behavioral evaluation was performed from the day after the spinal cord injury to the 20th day.
1. Basso, Beattie, and Bresnahan (BBB) scale
BBBscale (Basso, Beattie, Bresnahan, Experimental Neurology, 1996, Vol. 139, pp. 244-256), which is currently the most common scoring technique, is used to evaluate animals with spinal cord injury. That is, the hind limb function of the mouse in the cage was visually observed for 5 minutes and scored from 0 to 21 points based on the BBBscale.
2. Standing behavior The number of standing by the hind limbs in the cage was measured for 5 minutes.

[Immunohistochemical staining]
The mouse on the 21st day after the spinal cord injury treatment was anesthetized, and saline was injected from the left ventricle to remove blood, followed by infusion of 4% paraformaldehyde-PBS (PFA) and fixation under reflux. The spinal cord was removed and fixed overnight in 4% PFA. The spine was opened, the spinal cord was removed, and a 2 cm long thoracic vertebra was cut around the damaged site. Subsequent replacement with 10%, 20%, and 30% sucrose solutions was followed by embedding with an OCT compound, and a 12 μm thick sagittal slice was prepared with a cryostat and attached to a gelatin-coated slide glass.

  The periphery of the slice was surrounded by DAKO PEN (manufactured by Dako Japan Co., Ltd.), and 4% PFA was dropped and fixed for 30 minutes. For the primary antibody reaction, mouse anti-phosphorylated neurofilament-H (NF-H) monoclonal antibody (dilution ratio 1: 1000) (Clone SMI312, Sternberger Monoclonals, Lutherville, Md., USA), rabbit anti-myelin basic protein ( MBP) polyclonal antibody (dilution ratio 1: 100) (Chemicon, Temecula, CA, USA), rabbit anti-peripheral myelin protein (PMP) -22 polyclonal antibody (dilution ratio 1: 500) (Lab Vision, Fremont, CA) US) was used. After overnight reaction at 4 ° C., the secondary antibodies were Alexa Fluor 488-labeled goat anti-mouse IgG antibody and Alexa Fluor 568-labeled goat anti-rabbit IgG antibody (both dilution ratio 1: 200) (Molecular Probes, Carlsbad, CA) , USA). Double-fluorescence immunostained sample gray matter, white matter damage site and its front and back are magnified using a fluorescence microscope AX-80 (manufactured by Olympus Co., Ltd.), and the fluorescence image is acquired and positive for the antibody. The integrated value of the area was measured by ATTO Densitograph (manufactured by ATTO Corporation).

[Data analysis]
Data are expressed as mean ± standard error (sem). Significance test was performed by analysis of variance (ANOVA) or two way repeated measured ANOVA, and post hoc test was performed by Dunnett's test. The significance level was 5%.

[Experimental result]
Hereinafter, the obtained results will be described with reference to the illustrated graph and the micrograph of the tissue.
The number of individuals per group was 4-6.
It can be seen that the hindlimb dysfunction of the spinal cord injury model mouse (control group) is significantly lower than that of the Sham group, both by the BBB scale shown in FIG. 1 and by the number of rises by the hindlimb shown in FIG. On the other hand, in the Witanoside IV oral administration group of spinal cord injury model mice, it was found that hind limb dysfunction was significantly improved both in the determination by BBB scale and in the number of rises by the hind limb as compared with the control group.

Moreover, about the axon tissue shown in FIG. 3, it turns out that it is extended in the horizontal direction (NF-H positive) in the Sham group (refer FIG. 3A and FIG. 3G). Moreover, the expression of central myelin (MBP positive) was observed (see FIGS. 3D and 3G).
On the other hand, in the control group, it can be seen that the axonal tissue is torn, and in particular, the torn axon fibers are solidified at the damage center (see FIGS. 3B and 3H). In the control group, central myelin was eliminated (see FIGS. 3E and 3H).
On the other hand, in the oral administration group of Withanoside IV administered with the therapeutic agent for spinal cord injury of the present invention, no axonal fiber mass was observed in the center of the injury, and instead horizontal axon extension was observed as in the Sham group. (FIG. 3C). On the other hand, central myelin remained missing (see FIGS. 3F and 3I).

  Attempts were made to detect peripheral myelin, and there was a slight increase in peripheral myelin in the control group (see FIGS. 8E and 8H). On the other hand, it was found that peripheral myelin was increased in the woundanoside IV oral administration group of the present invention, particularly around the axon of gray matter (see FIGS. 8F and 8I).

  From the above results, it was clear that the repaired effect of the damaged site was observed in the oral administration group of the Witanoside IV of the present invention as compared with the control group.

As described above, it is possible to provide a therapeutic agent for spinal cord injury or a food / pharmaceutical composition that shows a simple and high effect by using the witanoside IV and its peripheral compounds according to the present invention.
Moreover, an effective treatment method for the purpose of treating spinal cords of humans or the like can be easily performed with the above-mentioned drug or food / pharmaceutical composition.

It is a score graph figure (until the 20th day after spinal cord injury) which shows determination of BBBscale of a mouse | mouth. ◯ indicates the Sham group, ● indicates the control group, and □ indicates the Witanoside IV administration group according to the present invention. It is a graph (up to the 20th day after spinal cord injury) of the number of rising by the hind limb in the cage of the mouse. ◯ indicates the Sham group, ● indicates the control group, and □ indicates the Witanoside IV administration group according to the present invention. A tissue of a thoracic spine section of a mouse 21 days after spinal cord injury was subjected to double fluorescent immunostaining using anti-phosphorylated NF-H monoclonal antibody (A, B, C) and anti-MBP polyclonal antibody (D, E, F). It is a micrograph. The left side is the head side, the right side is the caudal side, and the arrow indicates the crushing part. “Sham” indicates the Sham group, “Cont” indicates the control group, and “WS-IV” indicates the Witanoside IV administration group according to the present invention (similar descriptions in the following drawings have the same meaning). G, H, and I are photographs in which the magnification of observation is increased by superposing D on A, E on B, and F on C, respectively. It is a graph which shows the fluorescence intensity of the positive region of the gray matter with respect to an anti- phosphorylated NF-H monoclonal antibody. “Rostral” indicates the head side and “Caudal” indicates the caudal side. It is a graph which shows the fluorescence intensity of the positive region of the white matter with respect to an anti- phosphorylated NF-H monoclonal antibody. “Rostral” indicates the head side and “Caudal” indicates the caudal side. It is a graph which shows the fluorescence intensity of the gray matter and white matter positive area | region with respect to an anti-MBP polyclonal antibody. It is the microscope picture of the structure | tissue which carried out the double fluorescence immunostaining of the thoracic-vertebral section of the mouse | mouth 21st after spinal cord injury using the anti-phosphorylated NF-H monoclonal antibody (left) and the anti-MBP polyclonal antibody (right). W represents a white matter portion and G represents a gray matter portion. Thoracic vertebral sections of mice 21 days after spinal cord injury were double fluorescently immunostained using anti-phosphorylated NF-H monoclonal antibodies (A, B, C) and anti-PMP-22 polyclonal antibodies (D, E, F). It is a microscope picture of a structure | tissue. The left side is the head side, the right side is the caudal side, and the arrow indicates the crushing part. Sham represents a Sham group, Cont represents a control group, and WS-IV represents a Witanoside IV administration group according to the present invention. G, H, and I are photographs in which the magnification of observation is increased by superposing D on A, E on B, and F on C, respectively. It is a graph which shows the fluorescence intensity of the gray matter and white matter positive area | region with respect to an anti- PMP-22 polyclonal antibody. It is a microscope picture of the structure | tissue which carried out the fluorescence immunostaining of the thoracic spine section of the mouse | mouth 21st after spinal cord injury using the PMP-22 polyclonal antibody. W represents a white matter portion and G represents a gray matter portion.

Claims (4)

A therapeutic agent for spinal cord injury comprising at least one compound of the following structural formula 1 as an active ingredient.
Structural formula 1
(In the formula, R ₁ and R ₂ are each independently a hydrogen atom, a hydroxyl group, an alkyl group, or an alkoxy group, and R ₃ is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a sugar residue. ) A therapeutic agent for spinal cord injury comprising withanoside IV of the following structural formula 2 as an active ingredient.
Structural formula 2
  A food / pharmaceutical composition comprising the therapeutic agent for spinal cord injury according to claim 1 or 2 as an active ingredient and having an action of recovering spinal cord injury. A method for recovering spinal cord injury, comprising administering an effective amount of the therapeutic agent for spinal cord injury according to claim 1 or 2 or the food / pharmaceutical composition according to claim 3.