WO2007094166A1

WO2007094166A1 - Therapeutic agent for spinal cord injury

Info

Publication number: WO2007094166A1
Application number: PCT/JP2007/051458
Authority: WO
Inventors: Chihiro Tohda; Natsuki Nakayama; Masashi Yamada; Hiroto Suzuki
Original assignee: Meiji Dairies Corporation
Priority date: 2006-02-14
Filing date: 2007-01-30
Publication date: 2007-08-23
Also published as: JP2009102226A

Abstract

A drug for recovery from a spinal cord injury, characterized by containing as an active ingredient a compound represented by the following structural formula (1) or a pharmaceutically acceptable salt thereof: Structural formula 1 (wherein R1 and R2 each independently is hydrogen, hydroxy, alkyl, or alkoxy; and R3 is hydrogen, hydroxy, alkyl, alkoxy, or a sugar residue).

Description

Specification

Spinal cord injury treatment

Technical field

TECHNICAL FIELD [0001] The present invention relates to a therapeutic agent for spinal cord injury, and in particular, a therapeutic agent for spinal cord injury containing Withanoside IV and its peripheral compounds as an active ingredient, a food product containing the same, and spinal cord injury using the same. Concerning recovery methods.

Background art

[0002] 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 of the 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. Spine, spinal cord injury as well as ligament injury results. (Spine & spinal cord injury, [searched January 20, 2006], Internet http: ZZwww. Rd. M mtr. Or. Jp / ~ sumihosp / sekisonbun.htm>). In addition to trauma, there are spinal cord injuries and disc herniation.

[0003] When the spinal cord is damaged, the movement of the limbs may cause paralysis of the sensation or, in severe cases, even respiratory problems, depending on the method of damage, degree of damage, and site of damage. The higher the location of the injury, the greater the range of paralysis and the more severe the injury. For example, chest damage may result in lower body paralysis, and neck damage may result in paralysis of all extremities.

[0004] At present, therapies for spinal cord injury 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. It is. 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 some literatures denying the high-dose steroid 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 have been developed (Non-patent Documents 2 and 3). [0005] On the other hand, various studies are being made on therapeutic methods using drugs for neurological dysfunction diseases. For example, Patent Document 1 proposes a therapeutic agent containing as an active ingredient a substance that induces proliferation and activation of secreted substances from rod cells as a therapeutic agent for nerve damage such as spinal cord injury. .

[0006] Further, Patent Document 2, Patent Document 3, and Patent Document 4 propose oxazopylo-mouth quinolines, pyrroloquinolines, indolequinone derivatives and the like as nerve growth factor production promoters.

[0007] Non-Patent Document 1: Kronvall E. et al., Lakartidningen, June 2005, 13-26; 102 (24-25): 1887-8, 1890

Non-Patent Document 2: Leroy PH. Et al., Blood Rev., 2005 November, 1 9 (6), p. 321-331

Non-patent document 3: Dezawa M. et al., Curr Mol Med., 2005

November, 5 (7), p. 723- 732

Patent Document 1: Japanese Patent Application Laid-Open No. 2004-002412

Patent Document 2: Japanese Patent Laid-Open No. 06-009396

Patent Document 3: Japanese Patent Laid-Open No. 06-211660

Patent Document 4: Japanese Patent Laid-Open No. 07-118152

Disclosure of the invention

Problems to be solved by the invention

[0008] However, since the therapeutic agent and the treatment method shown in Patent Document 1 described above use rod-shaped cells or subsets thereof, they lack simplicity and are difficult to standardize. In addition, any of the other methods and therapeutic drugs described above are not fully established, but their effects are insufficient. Therefore, if the number of patients with spinal cord injury is increased and the background is taken into consideration, there is a strong demand for the development of a therapeutic method and Z or a therapeutic agent that has a simple, high and effective effect on spinal cord injury.

Accordingly, an object of the present invention is to provide a drug for recovering spinal cord injury that shows a simple and high effect, and a method for recovering spinal cord injury.

Means for solving the problem [0010] As a result of diligent 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, withanoside IV and its The present inventors have completed the present invention by finding out that peripheral compounds have a spinal cord injury recovery action.

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.

[0011] Therefore, as one embodiment of the present invention, there is provided a therapeutic agent for spinal cord injury comprising at least one compound of the following structural formula 1 as an active ingredient.

[0012] Structural Formula 1

(In the formula, R and R are each independently a hydrogen atom, a hydroxyl group, an alkyl group, or an

1 2

R is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a sugar residue

Three

It is. )

[0013] 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 as an active ingredient Uitanoside IV of the following structural formula 2.

[0014] Structural formula 2

[0015] Further, another aspect of the present invention is a food / pharmaceutical composition comprising the therapeutic agent for spinal cord injury as an active ingredient and having an action of recovering spinal cord injury.

[0016] Here, the food / pharmaceutical composition means a food composition or a pharmaceutical composition.

[0017] Still 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.

[0018] Further, as another aspect of the present invention, as described in any one of Structural Formula 1 and Structural Formula 2 for producing the therapeutic agent for spinal cord injury or the food / pharmaceutical composition, Use of a compound or a pharmaceutically acceptable salt thereof.

The invention's effect

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

[0020] In addition, an effective therapeutic method for the purpose of treating spinal cords such as humans can be provided simply by using the above-mentioned drug or a food / pharmaceutical composition containing an effective amount thereof.

Brief Description of Drawings

[0021] FIG. 1 is a score graph (up to 20th day after spinal cord injury) showing the determination of the BBB scale of mice. ◯ indicates the Sham group, 參 indicates the control group, and the mouth indicates the withanoside IV administration group according to the present invention.

[Fig. 2] A graph of the number of rises by the hind limbs in the cage of the mouse (up to 20 days after spinal cord injury). 〇 indicates the Sham group, 參 indicates the control group, and the mouth indicates the witanoside according to the present invention. The given group is shown.

[Fig. 3] Thoracic vertebral sections of mice 21 days after spinal cord injury were treated with anti-phosphate NF-H monoclonal antibody (A, B, C) and anti-MBP polyclonal antibody (D, E, F). It is a microscope picture of the tissue which carried out the heavy fluorescence immunostaining. The left side is the head side, the right side is the caudal side, and the arrow indicates the crushing part. “rsh am” indicates the Sham group, “Cont” indicates the control group, and “WS-IV” indicates the witanoside and 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 observation magnification is increased by superposing D on A, E on B, and F on C, respectively.

FIG. 4 is a graph showing the fluorescence intensity of a white matter positive region against anti-phosphate NF-H monoclonal antibody.

[5] FIG. 5 is a graph showing the fluorescence intensity of the gray matter positive region for anti-phosphate NF-H monoclonal antibody.

[6] Fig. 6 is a graph showing the fluorescence intensity of the gray matter and white matter positive regions for the anti-MBP polyclonal antibody.

[Fig. 7] Tissue from a thoracic spine section of a mouse on the 21st day after spinal cord injury, revealed by double fluorescent immunostaining using anti-phosphate NF-H monoclonal antibody (left) and anti-MBP polyclonal antibody (right) It is a microscopic photograph. W indicates the white matter part and G indicates the gray matter part.

[Fig.8] Thoracic vertebral sections of mice on the 21st day after spinal cord injury were treated with anti-phosphate NF-H monoclonal antibody (A, B, C) and anti-PMP-22 polyclonal antibody (D, E, F) 2 is a photomicrograph of tissue that has been double-fluorescent immunostained. The left side is the head side, the right side is the caudal side, and the arrow indicates the crushing part. Sham represents the Sham group, Cont represents the control group, and WS-IV represents the witanoside and 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.

FIG. 9 is a graph showing fluorescence intensities of gray matter and white matter positive regions against anti-PMP-22 polyclonal antibody.

FIG. 10 is a micrograph of a tissue obtained by fluorescent immunostaining a thoracic spine section of a mouse 21 days after spinal cord injury using a PMP-22 polyclonal antibody. W indicates the white matter portion and G indicates the gray matter portion. BEST MODE FOR CARRYING OUT THE INVENTION

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

[0023] As described above, in order to solve the above-described problems, the present invention provides a therapeutic agent for spinal cord injury comprising as an active ingredient at least one compound represented by the following structural formula 1.

[0024] Structural Formula 1

[0025] In Structural Formula 1, R and R are each independently a hydrogen atom, a hydroxyl group, or an

1 2

It is preferably a kill group or an alkoxy group. R is a hydrogen atom, a hydroxyl group, an alkyl group.

Three

It is preferably a group, an alkoxy group, or a sugar residue. R and R are particularly preferably

1 2

Each is independently a hydrogen atom or a hydroxyl group. R is particularly preferably

Three

It is a sugar residue.

[0026] Examples of the alkyl group as the substituent are not limited to these examples, but preferred examples include linear or branched alkyl groups having 1 to 6 carbon atoms, such as methyl Group, ethyl group, 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 a carbon number of 6 to 6, 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.

[0027] Examples of the sugar residue as the substituent are not limited to the present invention, but a monosaccharide, a sugar derivative, an oligosaccharide composed of 2-7 heptasaccharide or an oligosaccharide derivative isotropic glycosyl group is preferred. Better Yes. With respect to the bond, the present invention is not limited to these, but it may be an α bond or a j8 bond, and an α bond and an ι8 bond may be mixed. Specific examples of the sugar residue include, but are not limited to, the residues of monosaccharides such as glucose, galactose, mannose, fructose, fucose, and arabinose; glucuronic acid, ァ -acetylyldarcosamine Residues of sugar derivatives such as cetylgalatatosamine and sialic acid; maltose, maltotriose, manoletotetraose, manoletopentaose, manoletohexaose, manoletoheptaose, lactose, gentiobiose, xylobiose Residues of oligosaccharides composed of 2 to 7 sugars such as primebellose, melibiose, noretinoose, vicyanose, laminaribiose, cellobiose, cellotriose, panose, etc., and particularly preferably composed of glucose and dalcose It is a residue of an oligosaccharide consisting of 2 to 7 sugars. In addition, the hydroxyl groups at the 2, 3, 4, and 6 positions may be modified with, for example, a acetyl group, a malol group, a phosphate group, or a methyl group.

[0028] Among the compounds represented by Structural Formula 1, Uitanoside IV represented by Structural Formula 2 is

It can be particularly preferably used because of its high effect.

[0029] Structural formula 2

[0030] Withanoside IV and its peripheral compounds used as a therapeutic agent for spinal cord injury of the present invention can be isolated from the strength of Ashwagandha (root of Withania somnifera Dunal). If it is arranged, it can be isolated by the method described in Chem. Phar. Bull., 50, ρ760-765 (2002). In addition, chemical synthesis methods The synthesized method can be used, and the method is not particularly limited.

[0031] It can also be obtained as a metabolite of withanoside. For example, one of the compounds included in structural formula 1, sominone (sominone, R = H, R = OH, R = OH) is a itanoside

one two Three

It is a metabolite of IV in the body and can be obtained, for example, by the method described in Heterocycles, Vol. 34, p689-698 (1992). Specifically, it can be obtained by treating Uitanoside 'IV with an enzyme such as naringinase at pH 5.25, 37 ° C for 3 days, and separating and purifying it by partition high-speed liquid chromatography. it can.

[0032] The food composition of the present invention is particularly applicable to foods for patients with spinal cord injury. Spinal cord injury recovery treatment can be performed simply by ingesting it directly as a special-purpose food such as a food for specified health use containing the drug of the present invention or a functional food.

[0033] Specifically, when used as a food composition, various foods and drinks (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crust, prepared milk powder, liquid food, disease Food for infants, foods such as infant milk powder, foods such as milk powder for lactating women, nutritional foods, etc.) may be added and ingested. The active ingredient can be used as it is, or it can be used according to conventional methods in ordinary food compositions such as other foods and mixed with food ingredients. Moreover, also about the property, the state of the food / beverage products normally used, for example,

, Solid (powder, granule, etc.), paste, liquid or suspension.

[0034] Other ingredients are not particularly limited, but foods and drinks containing the drug of the present invention include water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavors. Etc. can be used as the main component. Proteins include, for example, whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, kappa zein, j8 —Lactoglobulin, α-lactalbumin, ratatopherin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof; butter, milky minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids And various milk-derived components such as lactose. Examples include sugars, processed starches (texturin, soluble starch, pre- starch starch, oxidized starch, starch ester, starch ether, etc.) and dietary fiber. Examples of lipids include lard, fish oil, etc., fractionated oils, hydrogenated oils, Animal fats and oils such as stealth exchange oil; and vegetable oils such as palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils and transesterified oils. Vitamins include, for example, vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, nonotic acid, piotin, inositol. , Choline, folic acid and the like, and examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium and the like. Examples of the organic acid include malic acid, citrate, lactic acid, and tartaric acid. These ingredients can be used in combination of two or more, using synthetic products and Z or foods rich in these.

[0035] The spinal cord injury therapeutic agent of the present invention The food / pharmaceutical composition comprising this as an active ingredient is determined in consideration of various factors such as the route of administration, age, weight, and symptoms of animals to be administered including humans. Can be set as appropriate. Although this invention is not limited to this, Preferably, 0.01-: LOO / z molZkgZday is suitable as an active ingredient.

The therapeutic agent and pharmaceutical composition for spinal cord injury of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, etc.).

[0036] Examples of the administration form of the spinal cord injury therapeutic agent and pharmaceutical composition according to the present invention include oral administration such as tablets, coated tablets, capsules, granules, powders, solutions, syrups, and emulsions. Can do. These various preparations are prepared according to the usual methods by using the therapeutic agent for spinal cord injury of the present invention as an excipient, binder, disintegrant, lubricant, coloring agent, flavoring agent, solubilizing agent, suspension agent, coating. It can be formulated using known auxiliaries that can be generally used in the pharmaceutical preparation technical field such as pharmaceuticals.

[0037] The amount of the spinal cord injury therapeutic agent of the present invention can be arbitrarily determined according to its purpose and use (pharmaceutical composition such as prophylactic agent and therapeutic agent). Although the present invention is not limited to this, the content is usually preferably 0.001 to 100% (wZw), and particularly preferably 0.1 to 100%, based on the total amount.

All prior art documents cited in this specification are incorporated herein by reference.

Example [0038] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

[0039] [Preparation of spinal cord injury model mouse]

The dorsal skin and muscle of a mouse (ddY, male, 6 weeks old) were dissected under ether anesthesia to expose the spine. The vertebral arch at levels 8-11 of the thoracic vertebra was then excised with an electric drill. A weight (2.5 g) was dropped 3 times on the exposed thoracic vertebra three times with a 2 cm vertical upward force, and the spinal cord was damaged. On the other hand, those treated up to laminectomy were designated as the Sham group.

At this time, the spinal cord injury model mouse was paralyzed in both hind limbs and could not move while supporting the body weight in the hind limbs. In addition, it was confirmed that the Sham group maintained normal motor function.

[0040] [Drug administration]

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

[0041] [Behavioral evaluation]

The following behavioral evaluation was performed from the day after spinal cord injury until the 20th day.

1. Basso, Beattie, and Bresnahan (BBB) scale

The most common scoring method currently used for the evaluation of animals with spinal cord injury was the BBB scale (Basso, Beattie, Bresnahan, Experimental Neurology, 1996, 139, p. 244-256). 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 BBB scale.

2. Stand-up behavior

The number of standing up by the hind limbs in the cage was measured for 5 minutes.

[0042] [Immunohistochemical staining]

Anesthetize the mouse on the 21st day after spinal cord injury treatment and inject saline from the left ventricle to remove it. The blood was then fixed and refluxed with 4% paraformaldehyde PBS (PFA). The spinal cord was removed together with the spine 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. After sequentially replacing with 10%, 20%, and 30% sucrose solutions, they were embedded with OCT compound, 12-m thick sagital si ice was prepared with a cryostat, and attached to a gelatin-coated slide glass.

[0043] The slice was surrounded by DAKO PEN (manufactured by Dako Japan Co., Ltd.), 4% PFA was added dropwise, and fixation was performed for 30 minutes. For the primary antibody reaction, mouse anti-phosphorylated neurofilament H (NF—H) monoclonal antibody (dilution ratio 1: 1000) (Clone SMI312, Stern berger Monoclonals, Luthervile, Marybund, USA), Rusa ³ rinmyelin basi c protein (MBP) polyclonal antibody (dilution ratio 1: 100) (Chemicon, Temecula, California, USA), rabbit anti-peripheral myelin protein (PMP) —22 polyclonal antibody (dilution ratio 1: 500) (Lab Vision, Fremont, California, USA). After 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, California, USA). Double-fluorescent immunostained sample gray matter, white matter damage site and its front and back are magnified using a fluorescence microscope AX-80 (manufactured by Olympus Corporation), and the fluorescence image is acquired and positive for antibody. The integrated value in the area was measured by ATTO Densitogmph (manufactured by Atoichi Co., Ltd.).

[0044] [Data analysis]

Data were expressed as mean standard error (s. E. M.). Significance test was performed by analysis of variance (ANO VA), two-way repeated measured ANOVA, and post hoc test was performed by Dunnett's test. The significance level was 5%.

[0045] [Experimental results]

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.

The hindlimb dysfunction in spinal cord injury model mice (control group) was measured according to the BBB scale shown in Fig. 1. Therefore, it can be seen that the number of rises by the hind limb shown in Fig. 2 is also significantly lower than that in the Sham group. On the other hand, in the group with spinal cord injury mice orally treated with orientated Vitanoside IV, it was found that hind limb dysfunction was significantly improved both in the BBB scale and in the number of rises by the hind limb compared to the control group. .

In addition, it can be seen that the axon tissue shown in FIG. 3 is horizontally extended (NF-H positive) in the Sham group (see FIGS. 3A and 3G). In addition, the expression of central myelin (MBP positive) was observed (see Fig. 3D and Fig. 3G).

On the other hand, in the control group, it can be seen that the axonal tissue is ruptured, and the ruptured axonal fibers are solidified particularly in the center of the damage (see FIGS. 3B and 3H). In the control group, central myelin was lost (see Figure 3E and Figure 3H).

On the other hand, in the oral administration group of Withanoside 'IV administered with the spinal cord injury therapeutic agent 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. (Figure 3C). On the other hand, central myelin remained missing (see Figure 3F and Figure 31).

[0047] When detection of peripheral myelin was attempted, peripheral myelin slightly increased in the control group (see FIGS. 8E and 8H). In contrast, in the oral administration group of Uitanoside 'IV of the present invention, peripheral myelin increased in the wound site, particularly around the gray axon (see FIG. 8F and FIG. 81). .

[0048] From the above results, it was apparent that the woundanoside 'IV oral administration group of the present invention had a repair action on the damaged site as compared with the control group.

Industrial applicability

[0049] As described above, it was possible to provide a therapeutic agent for spinal cord injury or a food product 'pharmaceutical composition that shows a simple and high effect by using Withanoside IV and its peripheral compounds according to the present invention.

In addition, an effective treatment method for the purpose of treating spinal cords of humans and the like can be easily performed using the above-mentioned drug or food / pharmaceutical composition.

Claims

The scope of the claims

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

1 2

Three

It is. )

[2] A therapeutic agent for spinal cord injury comprising as an active ingredient Uitanoside 'IV of the following structural formula 2.

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 a recovery action for spinal cord injury.

The therapeutic agent for spinal cord injury according to claim 1 or 2, or the food according to claim 3 A method for recovering spinal cord injury, which comprises administering an effective amount of the composition.

Use of a compound of the following structural formula 1 or a pharmaceutically acceptable salt thereof for the manufacture of a spinal cord injury therapeutic agent or a food / pharmaceutical composition;

Structural formula 1

1 2

R is a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group, or a sugar residue. ) ₍