Agent for improving dysfonction caused by spinal cord compression

Abstract

Provided is an agent for improving dysfunction caused by spinal cord compression, wherein said agent contains at least one selected from the group consisting of Cistanche deserticola, Acteoside, and Echinacoside.

Description

Agent for improving dysfunction due to spinal cord compression

[0001] The present invention relates to an agent for improving dysfunction due to spinal cord compression.

[0002] As those in which dysfunction occurs due to spinal cord compression, there are cervical spondylotic myelopathy, spinal canal stenosis, foraminal stenosis, intervertebral disc herniation, spondylolisthesis, ossification of the posterior longitudinal ligament, etc. Among these, for example, cervical spondylotic myelopathy causes symptoms such as clumsy hand movements, leg entanglement during walking, and numbness in the hands and feet due to spinal cord compression. The diagnosis of cervical spondylotic myelopathy is comprehensively performed by neurological findings, evaluation by the severity of the cervical JOA score (Japanese Orthopaedic Association Cervical Spondylosis Treatment Judgment Criteria) and JOACMEQ (Japanese Orthopaedic Association Cervical Spinal Cord Disorder Evaluation Questionnaire), and image diagnosis by X-ray and MRI.

[0003] Yoshimatsu et al., Orthopaedics and Traumatology Surgery (2000) 49: 1006 - 1010

[0004] As the standard treatment for cervical spondylotic myelopathy that has been carried out so far, when the compression of the spinal cord is severe by MRI diagnosis or when it is resistant to conservative treatment, decompression by surgical application is performed. In drug treatment among conservative treatments, anti-inflammatory analgesics, vitamin B12, muscle relaxants, anti-anxiety drugs, prostaglandin preparations, steroids, etc. are administered. However, there is no report that these drug therapies are effective for spinal cord symptoms.

[0005] Protective therapies such as wearing a cervical collar or cervical traction therapy may also be taken, but the evidence levels of these effects are not high. The prognosis in the case of remaining untreated is deterioration of symptoms due to trauma, etc., gradual deterioration, or rapid deterioration, and there is almost no spontaneous cure. Also, in conservative treatment, the improvement is 24%, unchanged is 14%, and deterioration is 62%, and it cannot be said that it is necessarily effective (Non-Patent Document 1). Thus, there has been no drug treatment that has been considered effective for cervical spondylotic myelopathy so far. In diseases presenting dysfunction due to spinal cord compression, there is a problem of dysfunction that is not improved or recurs even after performing surgical removal of the compression.

[0006] An object of the present invention is to provide a novel agent for improving dysfunction due to spinal cord compression.

[0007] The present invention relates, for example, to the following [1] to [3]: [1] An agent for improving functional impairment caused by spinal cord compression, comprising at least one selected from the group consisting of Cistanche tubulosa extract, acteoside, and echinacoside. [2] The agent for improving functional impairment according to [1], wherein the functional impairment is at least one selected from the group consisting of sensory dysfunction and motor dysfunction. [3] The agent for improving functional impairment according to [1] or [2], wherein the functional impairment caused by spinal cord compression is due to cervical spondylotic myelopathy, spinal stenosis, foraminal stenosis, herniated disc, spondylolisthesis, and / or ossification of the posterior longitudinal ligament.

[0008] The present invention provides a novel agent for improving functional impairment caused by spinal cord compression.

[0009] This graph shows the results of the grip strength test. *p<0.05, **p<0.01, sham surgery group vs. compression group, two-sided independent t-test. Values ​​are mean ± standard deviation. Sham surgery group: n=6 mice, compression group: n=8 mice. This graph shows the results of the grip strength test. †p<0.05, sham surgery / water group vs. compression / water group, interaction between days × group [F(5,75) = 2.54, p = 0.0354]. #p<0.05, compression / water group vs. compression / Cistanche extract group, drug effect [F(1,16) = 4.72, p = 0.0452], repeated measures two-way ANOVA. ****p<0.0001, post-hoc Bonferroni test. Sham surgery / water group: n=8 mice, compression / water group: n=9 mice, compression / Cistanche extract group: n=9 mice. The values ​​are mean ± standard deviation. This is a graph showing motor nerve density. *p < 0.05, versus compression / water group, one-way ANOVA, post-hoc Bonferroni test. Sham surgery / water group: n = 8 mice, compression / water group: n = 8 mice, compression / Cistanche extract group: n = 9 mice. The values ​​are mean ± standard deviation. α = 0.05, 1 - β = 0.7838, r = 65307. (a) is a graph showing the amount of acteoside per tissue that migrated to the spinal cord, and (b) is a graph showing the amount of echinacoside per tissue that migrated to the spinal cord. Cortex: n = 3, Spinal cord: n = 3. This is a graph showing the effect of continuous intraventricular administration of acteoside on grip strength in cervical spinal cord compression model mice. The 5th to 7th cervical spinal cords of mice were compressed with a microscrew to a depth of 2 mm for 1 minute. Seven days after compression, acteoside dissolved in artificial cerebrospinal fluid, or artificial cerebrospinal fluid alone, was continuously administered intraventrally for 14 days. Grip strength was measured every two days using an inclined wire mesh test. Sham surgery / artificial cerebrospinal fluid administration group n=6, compression / artificial cerebrospinal fluid administration group n=6, compression / acteoside administration group n=7. Mean ± standard deviation. Compression / artificial cerebrospinal fluid administration group vs. compression / acteoside administration group, day × drug interaction; (F(20,160) = 23.55, P < 0.0001), drug effect; (F(2,16) = 51.40, P < 0.0001). *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. compression / artificial cerebrospinal fluid administration group, repeated measures two-way ANOVA. Post-hoc Bonferroni test. This graph shows the effect of continuous intraventricular administration of acteoside on the forelimb grip strength of a cervical spinal cord compression model mouse. The fifth to seventh cervical spinal cords of the mice were compressed with a microscrew to a depth of 2 mm for 1 minute.Seven days after compression, acteoside dissolved in artificial cerebrospinal fluid, or artificial cerebrospinal fluid alone, was continuously administered intraventricularly for 14 days. Forelimb grip strength was measured every two days using a forelimb grip strength test. Sham surgery / artificial cerebrospinal fluid administration group n=6, compression / artificial cerebrospinal fluid administration group n=6, compression / acteoside administration group n=7. Mean ± standard deviation. Compression / artificial cerebrospinal fluid administration group vs. compression / acteoside administration group, day × drug interaction; (F(12,96) = 5.585, P < 0.0001), drug effect; (F(2,16) = 40.16, P < 0.0001). *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. compression / artificial cerebrospinal fluid administration group, repeated measures two-way ANOVA. Post-hoc Bonferroni test. This graph shows the change in VAS over time. Interaction between time and drug: F(2,74) = 2.532, P = 0.0864, ​​Drug effect: F(1.37) = 3.987, P = 0.0532. Cistanche extract group: n = 19, Placebo group: n = 20. *p < 0.05, vs. placebo group, repeated measures two-way ANOVA, post-hoc Bonferroni test. This graph shows the change in VAS before administration and after 24 weeks. Cistanche extract group: n = 19, Placebo group: n = 20, two-tailed test. This graph shows the change in trunk sensory impairment in the JOA score before administration and after 24 weeks. Cistanche extract group: n = 19, Placebo group: n = 20, Wilcoxon rank-sum test. This graph shows the change in lower limb motor function in the JOACMEQ from 12 to 24 weeks after administration. Cistanche extract group: n=19, placebo group: n=20, two-tailed test. This graph shows the change in VAS. The number of people who reported improvement, no change, or deterioration in VAS before administration, 12 weeks and 24 weeks after administration was compared between the Cistanche extract group and the placebo group. Cistanche extract group: n=19, placebo group: n=20, binomial test. This graph shows the time course of shoulder and elbow motor function in the JOA score after administration of Cistanche extract. Cistanche extract administration group: n=19, placebo administration group: n=20, repeated measures two-way ANOVA, interaction of time × Cistanche extract [F[2,74] = 3.405, P = 0.0385]; ## P < 0.05 This graph shows the time course of trunk sensory function in the JOA score after administration of Cistanche extract.Cistanche extract group: n=19, placebo group: n=20, repeated measures two-way ANOVA, time × Cistanche extract interaction [F[2,74] = 5.052, P = 0.0088]; ## P < 0.05.

[0010] The following describes in detail embodiments for carrying out the present invention, but the present invention is not limited to the following embodiments.

[0011] The agent for improving functional impairment caused by spinal cord compression according to this embodiment (hereinafter also referred to as the "improving agent") contains at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside as an active ingredient.

[0012] In this specification, "nikuchuyou" refers to the fleshy stem of Cistanche salsa, Cistanche deserticola, or Cistanche tubulosa. From the viewpoint of containing high concentrations of acteoside and echinacoside, "nikuchuyou" is preferably the fleshy stem of Cistanche tubulosa.

[0013] The Cistanche tubulosa may be used as is, or it may be treated by drying, freezing, crushing, or extraction. Preferably, the Cistanche tubulosa is an extract obtained by extracting Cistanche tubulosa.

[0014] The Cistanche tubulosa used for extracting the extract may be the fleshy stem as is, or the fleshy stem may have been dried, frozen, cut, or crushed. Water, ethanol, methanol, or a mixture thereof can be used as the extraction solvent for the Cistanche tubulosa extract. The extraction solvent is preferably a mixture of water and ethanol, preferably a mixture of 20-40% by mass of ethanol and 60-80% by mass of water, preferably a mixture of 25-35% by mass of ethanol and 65-75% by mass of water, and more preferably 30% ethanol - 70% water. Extraction may be carried out at room temperature (e.g., 20-30°C, 23-27°C) or under heating, for example, at 20-70°C, 30-65°C, 40-65°C, or 50-65°C. The extraction time may be, for example, 1 to 24 hours.

[0015] Cistanche extract may be obtained by further concentrating, diluting, drying, and / or powdering an extract of Cistanche. Drying methods may include, for example, room temperature drying or freeze-drying. Commercially available Cistanche extract may also be used.

[0016] Cistanche extract contains acteoside and echinacoside as its main physiologically active components. As shown in the examples described below, it has been confirmed that at least acteoside and echinacoside are transferred into the spinal cord in mice orally administered with Cistanche extract.

[0017] Acteoside and echinacoside may be of natural origin, such as from Cistanche tubulosa, or they may be synthetic products.

[0018] The improving agent according to this embodiment contains at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside as an active ingredient, and can therefore improve functional impairment caused by spinal cord compression.

[0019] Functional impairment due to spinal cord compression may include, for example, functional impairment due to cervical spondylotic myelopathy, spinal stenosis, foraminal stenosis, herniated disc, spondylolisthesis, and / or ossification of the posterior longitudinal ligament. These symptoms share the common characteristic of being spinal cord compression-related. Functional impairment due to spinal cord compression may include, for example, functional impairment due to cervical spinal cord compression.

[0020] Functional impairments due to spinal cord compression may include, for example, sensory impairment, motor impairment, and / or autonomic nervous system dysfunction. Sensory impairments may include, for example, the occurrence of pain, numbness, and / or hypersensitivity, and dulling of pain and touch sensation. Sensory and motor impairments may include, for example, impairments of sensory and motor function in the upper limbs (shoulders, arms, fingers), lower limbs (feet), waist, and / or neck.

[0021] Improvement of functional impairment may include, for example, treatment, alleviation, reduction in frequency, or prevention of worsening of functional impairment. Improvement of functional impairment also includes improvement in quality of life (QOL) as a result of the improvement of functional impairment.

[0022] The target population for the improving agent according to this embodiment may be patients with cervical spondylotic myelopathy, spinal stenosis, foraminal stenosis, herniated disc, spondylolisthesis, and / or ossification of the posterior longitudinal ligament, and may also be those who have sensory and / or motor dysfunction resulting from these symptoms. For example, the target population is more preferably those with a cervical JOA score of 8 or more and less than 15 points. Furthermore, the target population may be patients whose symptoms are not severe and who do not require early surgery, or patients who have already undergone surgery.

[0023] To date, there are no known technologies to improve functional impairment caused by spinal cord compression. Given the large number of patients suffering from functional impairment due to spinal cord compression and the lack of effective treatments, this invention represents a technology of great social significance.

[0024] The active ingredient in the improving agent according to this embodiment, which includes at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside, may be present in solid content of, for example, 0.1% or more, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, 99% or more, or 100% or more, relative to the total amount of the improving agent. The active ingredient in the improving agent according to this embodiment, which includes at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside, may be, in terms of solid content, 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 93% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 60% by mass or less, 55% by mass or less, 50% by mass or less, 45% by mass or less, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 8% by mass or less, 5% by mass or less, 3% by mass or less, or 1% by mass or less, relative to the total amount of the improving agent.

[0025] The dosage (ingestion amount, administration amount) of the improving agent according to this embodiment may be adjusted as appropriate depending on the severity of the disease / symptoms, age, sex, weight, administration method, specific type of disease, etc. For example, the amount of Cistanche tubulosa, acteoside, and echinacoside per day may be approximately 1 to 6000 mg / kg, 5 to 3000 mg / kg, or 10 to 1200 mg / kg in solid content.

[0026] The improving agent according to this embodiment may be administered in a single dose or in multiple doses. The administration method may be oral or parenteral. Examples of parenteral administration include rectal administration, nasal administration, pulmonary administration, and injection. Oral administration is preferred.

[0027] The improving agent according to this embodiment may be the active ingredient as is, or it may further contain other components in addition to the active ingredient. Examples of other components include those commonly used as raw materials for pharmaceutical formulations, such as carriers, excipients, binders, disintegrants, lubricants, coating agents, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, and antioxidants. These other components may be used individually or in combination of two or more.

[0028] Examples of carriers include animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resins; silicone oils; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethylcellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol, and sorbitol; sugars such as glucose and sucrose; inorganic powders such as anhydrous silicic acid, aluminum magnesium silicate, and aluminum silicate; and purified water.

[0029] Examples of excipients include lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide.

[0030] Examples of binders include polyvinyl alcohol, gelatin, methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyvinyl acetal diethylaminoacetate, and corn starch.

[0031] Examples of disintegrants include corn starch, low-substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate, croscarmellose sodium, calcium citrate, dextrin, pectin, and carboxymethylcellulose calcium.

[0032] Examples of lubricants include magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, and sucrose fatty acid esters. Examples of flavoring and deodorizing agents include cocoa powder, menthol, aromatic powders, peppermint oil, borneol, and cinnamon powder.

[0033] Examples of the forms (dosage forms) of the improving agent according to this embodiment include tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules, syrups, oral solutions, lozenges, jellies, inhalants, suppositories, injections, ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions, topical solutions, sprays, topical aerosols, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, rectal soft capsules, and the like.

[0034] The improving agent according to this embodiment may be a food or beverage, or an additive to a food or beverage.

[0035] Examples of food and beverages include general foods, health foods, foods for specified health uses, foods with nutritional function claims, supplements (nutritional supplements), animal feed, and food additives.

[0036] Food and beverages may be intended for specific target groups [for example, elderly people, patients or sick people (e.g., patients with cervical spondylotic myelopathy, spinal stenosis, foraminal stenosis, herniated discs, spondylolisthesis, and / or ossification of the posterior longitudinal ligament, and those with sensory and / or motor impairments resulting from these conditions)].

[0037] When at least one substance selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside is used as a food or beverage such as a health food or nutritional supplement, it can be prepared, for example, by conventional means, in the form of tablets, capsules (soft capsules, hard capsules, etc.), powders, granules, liquids (suspensions, syrups, etc.), emulsions, jellies, sticks, etc. Tablets include disintegrating tablets (orally disintegrating tablets).

[0038] Food and beverages may contain food additives (food-grade additives). Examples of food additives include excipients (e.g., wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, pregelatinized starch, casein, magnesium aluminosilicate, calcium silicate, etc.), binders (e.g., pregelatinized starch, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc.), disintegrants (e.g., cellulose, hydroxypropylcellulose, corn starch, etc.), and fluidizers (e.g., Examples include light anhydrous silicic acid, sucrose fatty acid esters, etc.), oils (e.g., vegetable oils such as soybean oil, sesame oil, olive oil, flaxseed oil, perilla oil, rapeseed oil, coconut oil, corn oil, or oils derived from animals or fish), nutrients (e.g., various minerals, various vitamins, amino acids), flavorings, sweeteners, flavorings, colorings, solvents (ethanol), salts, surfactants, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, thickeners, lubricants, encapsulating agents, suspending agents, coatings, and preservatives. Food additives may be used individually or in combination of two or more types.

[0039] When at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside is used as a food additive, the food and beverages include foods [for example, noodles (soba, udon, Chinese noodles, instant noodles, etc.), confectionery (candy, gum, chocolate, snacks (potato chips, etc.), biscuits, cookies, gummies, jelly, jam, butter, cream (cream puffs, etc.), cakes, etc.), bread, processed seafood or livestock products (fish cakes, ham, sausages, etc.)]. Examples include dairy products (processed milk, fermented milk, etc.), oils and fats and processed oils and fats (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc.), seasonings (sauces, dips, etc.), retort foods (curry, stew, rice bowls, porridge, rice gruel, etc.), frozen desserts (ice cream, sherbet, shaved ice, etc.), fried foods (croquettes, french fries, fried chicken, etc.), and beverages (tea beverages, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, etc.).

[0040] The amount of at least one substance selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside in food and beverages can be appropriately selected depending on the form of addition and administration, and may be, for example, 0.0001% by weight or more (e.g., 0.001 to 50% by weight), 0.003% by weight or more (e.g., 0.005 to 30% by weight), or 0.01% by weight or more (e.g., 0.05 to 10% by weight) in terms of solid content.

[0041] The improving agent according to this embodiment may be a pharmaceutical, quasi-drug, or food or beverage itself, or may be used as an ingredient in such products. Pharmaceuticals, quasi-drugs, and food or beverages containing the improving agent according to this embodiment as one ingredient can be manufactured, for example, by adding the improving agent to the raw materials or intermediate products of such products.

[0042] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0043] 1. Model Mouse Test (1) Administration of Cistanche Extract [Creation of Cervical Spinal Cord Compression Model Mice] Cervical spinal cord compression model mice were created by artificially compressing the cervical spinal cord. ddY mice (male, 9 weeks old) were used for the test. The mice were placed in a transparent plastic cage (23 × 16 × 12 cm) and raised in an environment with a 12-hour light-dark cycle (specified 7:00-19:00), constant temperature and humidity (23±2℃, 55±10%). Water and solid feed were provided ad libitum.

[0044] Three kinds of mixed anesthetics were intraperitoneally administered to place the mice under anesthesia, and the back of the neck was shaved with a razor. The skin was incised, and the muscles were separated with forceps to expose the spine, and the third to fifth cervical vertebrae were exposed. The third to fifth cervical vertebrae were resected to expose the third to sixth cervical spinal cords. A micro screw with a flat tip (diameter 1.4 mm, pitch 0.3 mm) was screwed into a nut (width of both sides 3 mm), and it was placed on the fourth to fifth cervical spinal cords with the bottom surface of the nut and the tip surface of the screw aligned. Dental cement and instant adhesive were used to fix the nut to the surrounding bone and muscle tissues, and it was left standing until the screw solidified. The solidified screw was rotated and pushed in to a depth of 1.5 mm. The incised muscles and skin were sutured, and an anti - sedative was intraperitoneally administered to wake the mice from anesthesia. During the operation and the waking period, the mice were placed on a hot plate to prevent hypothermia. The mice that underwent cervical spinal cord compression surgery were used in the test as the "compression group".

[0045] As a sham operation, among the above - mentioned cervical spinal cord compression surgeries, anesthesia, hair shaving, and exposure of the spine were performed, and then it was sutured without compression. The mice that underwent the sham operation were used in the test as the "sham operation group".

[0046] [Motor function evaluation] A grip strength test of the mice was performed using a traction meter (Brain Science Ideas). (Figs. 1, 2) The grip strength test evaluates the grip strength by placing the mouse on the wire mesh part of the traction meter, pulling the tail parallel to the wire mesh, and measuring the force required for the mouse to leave the wire mesh. Three measurements were taken for each mouse, and the average value was adopted.

[0047] [Drug Administration] As the Cistanche tubulosa extract, an extract obtained by extracting the fleshy stem of Cistanche tubulosa (produced in Xinjiang) with 30% ethanol - 70% water (Alps Pharmaceutical Co., Ltd.) was used. The content of acteoside in the extract was 6.2% by mass in terms of solid content, and the content of echinacoside was 27.8% by mass in terms of solid content. An extract preparation obtained by mixing the Cistanche tubulosa extract and excipients (dextrin, fine silicon dioxide) at a ratio of 1:1 was used in the experiment. The dosage for mice was 492 mg / kg / day as the Cistanche tubulosa extract (984 mg / kg / day as the extract preparation). Mice in the Cistanche tubulosa extract group were inoculated with the Cistanche tubulosa extract dissolved in sterilized water by drinking water. Mice in the water group were allowed to ingest the solvent (sterilized water) by drinking water.

[0048] [Quantification of Motor Neurons] (Preparation of Spinal Cord Tissue Sections) Under deep anesthesia by isoflurane inhalation, mice 63 days after the compression surgery were exsanguinated by perfusion of the heart with ice-cold physiological saline. Subsequently, the tissue was fixed by perfusion with an ice-cold 4% paraformaldehyde - PBS solution. The skin on the back of the neck was incised, and the first cervical spinal cord - seventh cervical spinal cord including the compressed cervical spinal cord was excised and immersed in 4% paraformaldehyde - PBS for 24 hours for further fixation. The cervical spinal cord was immersed in a 30% sucrose - PBS solution for replacement and then stored at -30°C. Using a cryostat (CM1860, Leica), 20-μm-thick sagittal serial sections of the cervical spinal cord were prepared and affixed to glass slides.

[0049] (Immunohistochemical staining of spinal cord sections) Spinal cord sections mounted on a glass slide were allowed to return to room temperature, and a border was drawn around the section on the glass slide with clear nail polish. First, the sections were treated with 0.5% Triton X-100-PBS solution for 5 minutes. The primary antibody reaction solution was prepared by adding mouse anti-NeuN antibody (#MAB377, Millipore, 500-fold dilution), goat anti-ChAT antibody (#AB144P, Millipore, 500-fold dilution), and normal donkey serum (final concentration 5%) to 0.5% Triton X-100-PBS solution. A secondary antibody reaction solution was prepared by adding Alexa Fluor 594-labeled donkey anti-mouse antibody (#A32744, Invitrogen, 1000-fold dilution) and Alexa Fluor 647-labeled donkey anti-goat antibody (#A32849, Invitrogen, 1000-fold dilution) to a 0.5% Triton X-100-PBS solution.

[0050] The primary antibody reaction solution was placed on the tissue section and reacted at 4°C for 24 hours. After removing the primary antibody reaction solution and washing, the secondary antibody reaction solution was added and reacted at room temperature for 2 hours. After two washes with PBS, the tissue was mounted using Aqua Poly Mout (Polyscience).

[0051] (Quantitative Analysis of Stained Images) A ​​fluorescent microscope BZ-X800 (Keyence Corporation) was used to observe the sections. For the quantification of the number of ChAT-positive motor neurons, three sections from the sagittal section of the spinal cord containing motor neurons were selected for quantification, and the quantification area was defined as a range extending 2 mm caudally from a point 0.5 mm away from the center of compression. The number of ChAT-positive cells within the area was measured and divided by the area of ​​the quantification area to calculate the number of motor neurons per unit area.

[0052] [Spinal Cortex Transfer of Major Components After Oral Administration of Cistanche Extract] ddY mice (male, 7 weeks old) were used. They were fasted for 18-20 hours prior to administration, and 10 g / kg of Cistanche extract dissolved in ultrapure water was orally administered. At 0.5, 1, 3, and 24 hours after administration, blood was removed by cardiac perfusion with ice-cold saline under deep anesthesia by isoflurane inhalation. The spinal cord was removed, homogenized with 10 times the volume of methanol, sonicated, and centrifuged at 12000 g for 10 minutes. The supernatant was collected in a tube and evaporated to dryness on a 50°C hot plate. 50% methanol was added to dissolve the solution, and it was centrifuged at 13000 g for 5 minutes. The supernatant was passed through a 0.45 μm filter and used as a sample for LC-MS. Using the samples prepared in this manner, acteosides and echinacosides in spinal cord tissue were quantified using an Accela HPLC system (Thermo Fisher Scientific) and an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific).

[0053] [Experimental Results] Figure 1 shows the grip strength of mice in the compression group and the sham surgery group after compression surgery. A decrease in grip strength occurred as early as 7 days after cervical spinal cord compression and continued to be observed until at least 28 days after compression.

[0054] Seven days after cervical spinal cord compression, Cistanche extract or water was administered orally. Grip strength tests were performed every seven days to evaluate the effect of the drug over the entire administration period. The progress after drug administration is shown in Figure 2. Compared to the control group (sham surgery group), the compression / water group showed a significant decrease in grip strength (number of days elapsed × drug administration, interaction: F(5,75) = 2.54, p = 0.0354). Compared to the compression / water group, the compression / Cistanche extract group showed a significant improvement in grip strength (drug administration effect: F(1,16) = 4.72, p = 0.0452).

[0055] The cervical spinal cord was removed 63 days after spinal cord compression (56 days after drug administration), sagittal sections were prepared, and the number of motor neurons in the region 1 mm caudal to the compression site was quantified. The results are shown in Figure 3. Compared to the sham surgery group, the compression / water group showed a significant decrease in the number of motor neurons. Compared to the compression / water group, the compression / Cistanche extract group had a significantly higher number of motor neurons (Figure 3).

[0056] The main components of Cistanche tubulosa extract are acteoside and echinacoside. Therefore, we investigated whether these components crossed into the spinal cord after oral administration of Cistanche tubulosa extract to mice. The results are shown in Figure 4. Acteoside and echinacoside were found to reach the spinal cord, peaking 0.5 hours after oral administration of the extract.

[0057] (2) Acteoside administration [Preparation of cervical spinal cord compression model mice] ddY mice (female, 12-13 weeks old) were used and reared in the same manner as in (1). The mice were anesthetized by intraperitoneal administration of a triple-compound anesthetic, and the posterior neck was shaved with a razor. The skin was incised, and the muscles were clamped aside to expose the spine. The 4th to 6th cervical vertebrae were resected, exposing the 5th to 7th cervical spinal cords. A flat-tipped micro-screw (1.4 mm in diameter) was attached to the tip of a manipulator mounted on a cerebrospinal cord fixation device, placed directly above the 5th to 7th cervical spinal cords, and pushed in to a depth of 2 mm for 1 minute. The incised muscles and skin were sutured, and Antisedan was administered intraperitoneally to awaken the mice from anesthesia. During surgery and during the recovery period, the mice were placed on a hot plate to prevent hypothermia. Mice that underwent cervical spinal cord compression surgery were used in the test as the "compression group". As a sham surgery, the procedure involved anesthesia, shaving, and spinal exposure, as described above for cervical spinal cord compression surgery, but without compression, and the area was sutured. Mice that underwent the sham surgery were used in the experiment as the "sham surgery group."

[0058] [Motor Function Assessment] A grip strength test was conducted on mice using a traction meter (Brain Science Idea Co., Ltd.). The grip strength test was performed by placing the mouse on the wire mesh of the traction meter, pulling its tail parallel to the mesh, and measuring the force required until the mouse released the mesh. Three measurements were taken for each mouse, and the average value was used.

[0059] To functionally evaluate the grip strength of the forelimbs and hindlimbs, an inclined wire mesh test was performed (Figure 5). A wire mesh (1.3 cm grid, 20 cm x 32 cm) was propped up at a height of 45 cm from the floor at an inclination of 30°, and mice were placed on the wire mesh and observed for 30 seconds to see if they fell. If a mouse did not fall, the angle of the wire mesh was increased by 10° and the same trial was repeated. This was continued until the angle of the wire mesh reached a maximum of 150°. At each angle, points were awarded as follows: fall: 0 points, not fall but not move: 1 point, not fall but move left, right, up, or down: 2 points. The total score for all successful angles was calculated. The maximum score was 26 points.

[0060] To measure grip strength specifically in the forelimbs, a forelimb grip strength test was performed (Figure 6). Weights were attached to steel wool. The number of weights was increased, and the maximum weight that could be held by both forelimbs was quantified.

[0061] [Drug Administration] Acteoside was continuously infused into the ventricles of the cerebral ventricles of a cervical spinal cord compression model mouse starting 7 days after compression. The infused acteoside was dissolved in artificial cerebrospinal fluid, with the concentration in the cerebrospinal fluid set to 10 μM at all times, and administered for 14 days using a microosmotic pump (infusion rate 0.25 μl / h, Alzet).

[0062] [Experimental Results] In the inclined wire mesh test, the scores in the model mice gradually increased until day 7 after compression. However, in the control group, which was administered only artificial cerebrospinal fluid to the model mice, the scores plateaued and did not change. On the other hand, in the acteoside intraventricular administration group, the scores began to increase from day 2 after administration, and in post-hoc Bonferroni tests, the scores increased significantly from day 6 after administration (Figure 5). Repeated measures two-way ANOVA showed a significant interaction (F(20,160) = 23.55, P < 0.0001) and a significant drug effect (F(2,16) = 51.40, P < 0.0001) between the acteoside administration group and the artificial cerebrospinal fluid-only administration group.

[0063] In the forelimb grip strength test, the control group of model mice administered only artificial cerebrospinal fluid from day 9 after compression showed no change in scores, remaining at a plateau. On the other hand, in the acteoside intracerebrospinal administration group, scores began to increase from day 4 after administration, and a significant increase in scores was observed at day 12 after administration in a post-hoc Bonferroni test comparison between the groups (Figure 6). Repeated measures two-way ANOVA showed a significant interaction (F(12,96) = 5.585, P < 0.0001) and a significant drug effect (F(2,16) = 40.16, P < 0.0001) between the acteoside administration group and the artificial cerebrospinal fluid-only administration group. In model mice with motor impairment in the forelimbs and hindlimbs due to cervical spinal cord compression, intracerebrospinal administration of acteoside significantly improved forelimb grip strength, and significant improvements were also confirmed in tests measuring forelimb and hindlimb grip strength.

[0064] 2. Human Clinical Trials [Subjects] Patients who met all of the following selection criteria and did not meet any of the following exclusion criteria were enrolled in the clinical trial. (Selection Criteria) 1) Age 40 to 85 years old on the date of consent. 2) Able to participate in examinations performed at Toyama University Hospital. 3) Meets the diagnostic criteria for cervical spondylotic myelopathy based on physical examination. 4) Cervical spine JOA score at screening is approximately 8 or higher and 15 or lower. Basically, patients are to be enrolled before surgery, but post-surgery enrollment is also possible if the JOA score is appropriate. 5) Consent to provide blood samples for clinical testing. 6) Able to take the study drug and have the patient or their family manage the administration of the study drug. 7) Consent not to make any extreme changes to lifestyle habits such as exercise habits and dietary habits during the study period.

[0065] (Exclusion Criteria) 1) Does not consent to participate in the clinical trial. 2) Has had an uncontrolled and clinically problematic health condition (diabetes, hypertension, thyroid / endocrine disorder, congestive heart failure, angina pectoris, heart disease, cancer, etc.), gastrointestinal disorder, dialysis or renal dysfunction, etc. within 3 months of consent. 3) Has a history of alcohol and drug abuse. 4) Is currently participating in another interventional clinical trial. 5) Has a cervical spine JOA score of 16 or higher at screening and has very mild cervical spondylotic myelopathy. 6) Has a cervical spine JOA score of 7 or lower at screening and has severe cervical spondylotic myelopathy that is judged to require early surgery. 7) For any other reason that the research staff deems unsuitable as a subject.

[0066] The attributes of the subjects in the clinical trial are shown in Table 1. Among the 19 subjects randomly assigned to the Cistanche extract group and the 20 subjects randomly assigned to the placebo group, there were no significant differences in age or JOA score, nor were there any significant differences in gender bias between the groups.

[0067]

[0068] [Test Drug] The same Cistanche extract preparation used in the mouse experiment was employed. The dose administered to humans was 2400 mg / person / day as Cistanche extract (4800 mg / person / day as the extract preparation).

[0069] [Method of administration] In the placebo group, participants took one packet of placebo (a mixture of dextrin, fine silicon dioxide, and coloring agent) once a day. In the Cistanche extract group, participants took one packet of Cistanche extract preparation once a day. Both the placebo and Cistanche extract groups took the medication for 24 weeks. Neither the researchers nor the participants knew which was the placebo or the test drug (double-blind study).

[0070] [Primary Endpoint] (VAS) The Visual Analogue Scale (VAS) is one of the most frequently used assessment scales in research on spinal and neurological disorders, including the cervical spine. It is a subjective assessment method by patients themselves, quantifying the degree of pain and numbness on a scale from 0 to 10. Here, the degree of pain and numbness experienced by patients was evaluated regardless of the location. Changes in VAS scores were calculated before administration, at 12 weeks (3 months) and 24 weeks (6 months) after administration, and compared between groups. The results are shown in Figure 7.

[0071] A repeated measures two-way ANOVA showed a significant drug effect between the Cistanche extract group and the placebo group (F(1,37) = 3.987, P = 0.0532). A significant trend in the interaction between time and the drug was also observed (F(2,74) = 2.532, P = 0.0864). Post-hoc Bonferroni tests showed that VAS scores significantly improved in the Cistanche extract group at 12 and 24 weeks after administration. A comparison of the change in VAS between pre-administration and 24 weeks after administration showed that the Cistanche extract group demonstrated improvement compared to the placebo group (Figure 8).

[0072] (Cervical Spine JOA Score) The Cervical Spine JOA score (Japanese Orthopedic Surgery Cervical Spondylosis Treatment Assessment Criteria) evaluates a total of seven items: fine motor skill impairment of the fingers, motor function (upper and lower limbs), sensory impairment (upper limbs, trunk, and lower limbs), and bladder and bowel dysfunction. The JOA score was analyzed for each of the seven lower items. As a result, in a comparison of the change in trunk sensory impairment before administration and 24 weeks after administration, the Cistanche extract group showed improvement compared to the placebo group (Figure 9).

[0073] (JOACMEQ) The JOACMEQ (JOA Cervical Myelopathy Evaluation Questionnaire) is a questionnaire in which patients subjectively answer questions about cervical spine function, upper limb function, lower limb function, bladder function, QOL, and VAS. Patients answer questions about their condition over the past week or so.

[0074] In the lower limb motor function items of the JOACMEQ, a comparison of the changes from 12 to 24 weeks after administration showed that the Cistanche extract group demonstrated a significant improvement compared to the placebo group (Figure 10). In the VAS, the difference between the number of people who improved and the number of people who worsened compared to 12 weeks after administration and compared to 24 weeks after administration showed that the Cistanche extract group had a significantly larger number of people who improved, while there was no difference in the placebo group (Figure 11). The questions regarding lower limb motor function in the JOACMEQ are shown in Table 2.

[0075]

[0076] In the 24-week time course of the sub-items of the JOA score, significant interactions were observed between the Cistanche extract group and the placebo group in shoulder and elbow motor function (Figure 12) and trunk sensory function (Figure 13).

Claims

1. An agent for improving functional impairment caused by spinal cord compression, comprising at least one selected from the group consisting of Cistanche tubulosa, acteoside, and echinacoside.

2. The corrective agent according to claim 1, wherein the functional impairment is at least one selected from the group consisting of sensory functional impairment and motor functional impairment.

3. The corrective agent according to claim 1 or 2, wherein the functional impairment due to spinal cord compression is caused by cervical spondylotic myelopathy, spinal stenosis, foraminal stenosis, herniated disc, spondylolisthesis, and / or ossification of the posterior longitudinal ligament.

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