Promoter of n-acetylgalactosaminyltransferase expression containing extract from inflamed tissue inoculated with vaccinia virus

Abstract

An object of the present invention is to provide an N-acetylgalactosaminyltransferase expression promoter containing an extract from inflamed tissues inoculated with vaccinia virus.SOLUTION: In the present invention, it was shown that the extract from inflamed tissues inoculated with vaccinia virus promotes the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells. Therefore, the extract from inflamed tissues inoculated with vaccinia virus or a preparation containing the same is useful as an agent for promoting the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel medicinal uses of an extract from inflamed tissue inoculated with vaccinia virus (hereinafter sometimes referred to as "the extract"), and more specifically to an N-acetylgalactosaminyltransferase (GalNAcT) expression promoter containing the extract. [Background technology]

[0002] Intervertebral discs are composed of three tissues with distinct biochemical and physical properties: the nucleus pulposus, the annulus fibrosus, and the cartilaginous endplates. The abundant proteoglycans in the nucleus pulposus retain a large amount of water, resulting in an intervertebral disc that is approximately 80% water. Disc degeneration is one of the major causes of lower back pain and can lead to the development of conditions such as disc herniation and spinal stenosis. Age-related disc degeneration varies significantly from person to person, but it is believed that changes in the constituent cells of the nucleus pulposus, resulting in matrix changes, and subsequent breakdown of the annulus fibrosus structure are strongly related to disc degeneration. In humans aged 60 and older, the proteoglycan content in the intervertebral disc declines from 65% of that in teens to approximately 30%, less than half of that, resulting in a decrease in the water content of the disc. This increases the susceptibility of discs to structural breakdown due to external factors.

[0003] Proteoglycans are compounds in which multiple polysaccharides (glycosaminoglycans, GAGs) consisting of consecutive disaccharide units such as chondroitin sulfate are bound to a tetrasaccharide linkage region in which carbohydrates are bound to serine residues in a core protein. Proteoglycans, which have diverse functions as biological components, are present in the extracellular matrix and cell surfaces of various major organs, the brain, skin, and tissues throughout the body, as well as being a major component of cartilage.

[0004] GAGs are classified into four types based on their disaccharide backbone: (1) heparin, heparan sulfate, (2) chondroitin sulfate, dermatan sulfate, (3) keratan sulfate, and (4) hyaluronic acid. These backbones undergo various modifications, primarily sulfation. In many cases, GAGs exist bound to core proteins, i.e., as proteoglycans. It is known that various glycosyltransferases and sulfotransferases are involved in the biosynthesis of GAGs.

[0005] Chondroitin sulfate is a type of GAG and is abundant as a side chain of aggrecan, a major component of the extracellular matrix of cartilage. It is known that chondroitin sulfate is reduced in cartilage from patients with degenerative disc disease and osteoarthritis (OA).

[0006] Chondroitin sulfate is a linear sulfated sugar chain composed of alternating disaccharide units, N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcUA). Two N-acetylgalactosaminyltransferases are involved in the biosynthesis of the chondroitin sulfate backbone. One is N-acetylgalactosaminyltransferase 1 (GalNAcT1), which transfers GalNAc to the terminal GlcUA residue of the linkage region. The other is N-acetylgalactosaminyltransferase 2 (GalNAcT2), which transfers GalNAc to the terminal GlcUA residue of the disaccharide repeating structure. Chondroitin sulfate biosynthesis begins with GalNAc being transferred to the terminal GlcUA residue of the linkage region by GalNAcT1. Subsequently, GlcUA is transferred alternately by glucuronyltransferase, and then GalNAc is transferred alternately by GalNAcT2, synthesizing the disaccharide repeating region and elongating the sugar chain. Finally, the constituent sugars are sulfated by sulfotransferases to form chondroitin sulfate. Thus, it has been revealed that in the biosynthesis of chondroitin sulfate, GalNAcT1 is involved in the initiation of sugar chain synthesis, and GalNAcT2 is involved in the elongation of the sugar chain. In other words, GalNAcT1 is involved in increasing the number of sugar chains, rather than in the elongation of the sugar chains of chondroitin sulfate.

[0007] The extract from inflammatory tissue inoculated with vaccinia virus (the present extract) contained in the N-acetylgalactosaminyltransferase expression promoter of the present invention or a preparation containing the same has analgesic, sedative, anti-stress, anti-allergic, immunostimulatory, anti-cancer, anti-cirrhosis, therapeutic effect on idiopathic thrombocytopenic purpura, therapeutic effect on postherpetic neuralgia, cerebral edema, dementia, spinocerebellar degeneration, etc., therapeutic effect on Raynaud's syndrome, diabetic neuropathy, sequelae of SMON, etc., inhibitory effect on kallikrein production, amelioration of peripheral circulatory disorders, amelioration of bone atrophy, and effects on sepsis and endotoxin. The extract is known to have a wide range of effects, including inhibiting nitric oxide production (effective in treating steroid shock), therapeutic effects against osteoporosis, therapeutic effects against AIDS due to its inhibitory effects on Nef activity and chemokine production, therapeutic effects against ischemic diseases such as cerebral infarction, therapeutic effects against fibromyalgia, therapeutic effects against infectious diseases, preventive or alleviating peripheral neuropathy caused by anticancer drugs, therapeutic effects against chronic prostatitis, interstitial cystitis and / or dysuria, promoting the production of neurotrophic factors such as BDNF, hepatoprotective effects, promoting the migration of pluripotent stem cells (Muse cells), and preventive or therapeutic effects against muscle damage. In addition, the extract or a preparation containing the extract is known to promote the synthesis of collagen and proteoglycan in chondrocytes (see Patent Document 1). However, it has not been previously known that the extract or a preparation containing the extract promotes GalNAcT expression or promotes GalNAcT1 expression more strongly than GalNAcT2. The fact that GalNAcT1 expression is more strongly promoted than GalNAcT2 indicates that its effect on increasing the number of sugar chains is stronger than the effect of chondroitin sulfate on sugar chain elongation, and no drug with such an effect has been known to date. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2012 / 051173 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a GalNAcT expression promoter and the like containing the extract. [Means for solving the problem]

[0010] As a result of extensive research into the pharmacological effects of this extract, the present inventors discovered that this extract has an excellent GalNAcT expression promoting effect, leading to the completion of the present invention. [Effects of the Invention]

[0011] Because the extract promotes GalNAcT expression, a formulation containing the extract can be an excellent therapeutic or preventive agent for diseases associated with intervertebral disc degeneration and OA. In particular, the extract has been used for many years as a highly safe drug with few side effects and other problems, making the present invention extremely useful. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is an electrophoretic diagram showing the results of Western blotting to examine the expression level of CSGALNACT1 protein in nucleus pulposus cells treated with the extract of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] This extract contains a non-protein active substance extracted and separated from the inflamed tissue of animals that have been inoculated with vaccinia virus and developed smallpox. This extract is liquid when extracted, but can also be made solid by drying. This preparation is extremely useful as a pharmaceutical. A specific product manufactured and sold in Japan by the applicant as this preparation is a "preparation containing extract of inflamed skin of rabbits inoculated with vaccinia virus" (trade name: NEUROTROPIN [registered trademark]) (hereinafter referred to as "NEUROTROPIN"). NEUROTROPIN is available in injections and tablets, both of which are ethical drugs.

[0014] The indications for Neurotropin injection are "lower back pain, cervicobrachial syndrome, symptomatic neuralgia, itching associated with skin diseases (eczema, dermatitis, hives), allergic rhinitis, and cold sensation, abnormal sensation, and pain associated with SMON (Subcutaneous myelopathy). The indications for Neurotropin tablets are "postherpetic neuralgia, lower back pain, cervicobrachial syndrome, periarthritis of the shoulder, and osteoarthritis." This formulation was created and developed by the applicant as a pharmaceutical product, and has been highly acclaimed for its excellent efficacy and safety, having been sold for many years and establishing a solid position in the Japanese pharmaceutical market.

[0015] The extract from inflammatory tissue inoculated with vaccinia virus according to the present invention can be obtained by disrupting inflamed tissue that has been inoculated with vaccinia virus and developed smallpox, adding an extraction solvent to remove tissue debris, deproteinizing the tissue, adsorbing the resulting tissue onto an adsorbent, and then eluting the active ingredient, for example, by the following steps. (A) Skin tissues of rabbits, mice, etc. that have been inoculated with vaccinia virus and developed smallpox are collected, the smallpox-affected tissues are crushed, and an extraction solvent such as water, phenol water, saline, or phenol-glycerin water is added, followed by filtration or centrifugation to obtain an extract (filtrate or supernatant). (B) The extract is adjusted to an acidic pH and heated to remove proteins, and the deproteinized solution is then adjusted to an alkaline pH and heated, after which it is filtered or centrifuged. (C) The obtained filtrate or supernatant is acidified and adsorbed onto an adsorbent such as activated carbon or kaolin. (D) An extract from inflammatory tissue inoculated with vaccinia virus can be obtained by adding an extraction solvent such as water to the adsorbent, adjusting the pH to alkaline, and eluting the adsorbed components. Thereafter, if desired, the eluate can be evaporated to dryness under reduced pressure or lyophilized to a dry product.

[0016] Various animals that can be infected with vaccinia virus, such as rabbits, cattle, horses, sheep, goats, monkeys, rats, and mice, can be used as animals for inoculating vaccinia virus to obtain inflamed tissue. The preferred inflamed tissue is inflamed skin tissue from rabbits. Any rabbit belonging to the order Lagomorpha can be used. Examples include European rabbits, domestic rabbits (domesticated European rabbits), hares (Japanese hare), pikas, and mountain hare. Of these, domestic rabbits are preferred. In Japan, domestic rabbits, which have long been bred and widely used as livestock or experimental animals, are also known as domestic rabbits. There are many breeds of domestic rabbits, but breeds such as the Japanese White and the New Zealand White can be used.

[0017] The vaccinia virus may be of any strain, including, for example, the Lister strain, the Dairen strain, the Ikeda strain, the EM-63 strain, and the New York City Board of Health strain.

[0018] More specifically, the basic extraction steps (A) to (D) of the present extract can be carried out, for example, as follows. Regarding process (A) Inflamed skin tissue is collected from rabbits after intradermal inoculation with vaccinia virus to induce smallpox. The collected skin tissue is washed and disinfected with a phenol solution or similar. The inflamed skin tissue is then disrupted and a 1- to 5-fold volume of extraction solvent is added. "Disruption" refers to the process of finely grinding the tissue into mincemeat using a mincer or similar device. The extraction solvent can be distilled water, physiological saline, or a weakly acidic or weakly basic buffer solution. Sterilizing agents and preservatives such as phenol, stabilizers such as glycerin, and salts such as sodium chloride, potassium chloride, and magnesium chloride may also be added as appropriate. Cellular tissue can also be disrupted to facilitate extraction by treatments such as freeze-thawing, ultrasound, cell membrane lytic enzymes, or surfactants. The resulting suspension is left to stand for 5 to 12 days. During this time, it may be heated to 30 to 45°C with or without stirring. The resulting liquid is then subjected to solid-liquid separation (filtration, centrifugation, etc.) to remove tissue debris, yielding a crude extract (filtrate or supernatant).

[0019] Regarding process (B) The crude extract obtained in step (A) is subjected to a deproteinization treatment. Deproteinization can be carried out by a commonly used, well-known method, such as heat treatment, treatment with a protein denaturant (e.g., acid, base, urea, guanidine, organic solvents such as acetone), isoelectric precipitation, salting out, etc. Next, a filtrate or supernatant is obtained from which the precipitated insoluble protein has been removed by a conventional method for removing insoluble matter, such as filtration using filter paper (cellulose, nitrocellulose, etc.), glass filters, Celite, Seitz filter plates, etc., ultrafiltration, centrifugation, etc.

[0020] Regarding process (C) The filtrate or supernatant obtained in step (B) is acidified, preferably to a pH of 3.5 to 5.5, and then subjected to adsorption onto an adsorbent. Examples of adsorbents that can be used include activated carbon and kaolin. The active ingredient can be adsorbed onto the adsorbent by adding the adsorbent to the extract and stirring, or by passing the extract through a column packed with the adsorbent. When the adsorbent is added to the extract, the solution can be removed by filtration, centrifugation, or the like, to obtain an adsorbent with the active ingredient adsorbed.

[0021] Regarding process (D) To elute (desorb) the active ingredient from the adsorbent obtained in step (C), an elution solvent is added to the adsorbent, adjusted to a basic pH, preferably between 9 and 12, and eluted at room temperature or by appropriate heating or stirring. The adsorbent is then removed by a conventional method, such as filtration or centrifugation. The elution solvent used can be a basic solvent, such as water, methanol, ethanol, isopropanol, or an appropriate mixture thereof, adjusted to a basic pH. Water adjusted to a pH between 9 and 12 is preferred. The amount of elution solvent can be appropriately selected. The pH of the eluate thus obtained can be adjusted to near neutral, or an appropriate mixture of these can be used as a drug substance, to finally obtain an extract from inflamed rabbit skin inoculated with vaccinia virus (the present extract).

[0022] Since this extract is liquid when it is produced, it can be appropriately concentrated or diluted to the desired concentration. When preparing formulations from this extract, heat sterilization is preferred. To prepare an injectable solution, for example, sodium chloride can be added to prepare a solution isotonic with physiological saline. While oral administration is possible in liquid or gel form, solid oral formulations such as tablets can also be prepared by subjecting the extract to appropriate procedures such as concentration and drying. Specific methods for preparing such solid oral formulations from this extract are described in the specifications of Japanese Patent Nos. 3818657 and 4883798. The resulting injectable or oral formulations are examples of this formulation.

[0023] Below, we will show an example of a method for producing this extract, as well as the results of pharmacological tests on the novel pharmacological action of this extract and its ability to promote the expression of N-acetylgalactosaminyltransferase, but the present invention is not limited in any way by the descriptions in these examples. [Example]

[0024] Example 1: Preparation of the extract Vaccinia virus was intradermally inoculated into the skin of healthy adult rabbits, and the affected skin was excised and collected. The collected skin was washed and disinfected with phenol solution, after which excess phenol solution was removed, and the tissue was crushed, mixed with phenol solution, and left for 3-7 days, after which it was heated to 35-40°C with stirring for an additional 3-4 days. The extract obtained by solid-liquid separation was then adjusted to pH 4.5-5.2 with hydrochloric acid, heated at 90-100°C for 30 minutes, and then filtered to deproteinize. The filtrate was then adjusted to pH 9.0-9.5 with sodium hydroxide, heated at 90-100°C for 15 minutes, and then subjected to solid-liquid separation.

[0025] The resulting deproteinized liquid was adjusted to pH 4.0-4.3 with hydrochloric acid, and activated carbon was added in an amount 2% of the deproteinized liquid mass. The mixture was stirred for 2 hours, followed by solid-liquid separation. Water was added to the collected activated carbon, and the pH was adjusted to 9.5-10 with sodium hydroxide. The mixture was stirred at 60°C for 90-100 minutes, then centrifuged to obtain the supernatant. Water was added again to the activated carbon precipitated by centrifugation, and the pH was adjusted to 10.5-11 with sodium hydroxide. The mixture was stirred at 60°C for 90-100 minutes, then centrifuged to obtain the supernatant. Both supernatants were combined and neutralized with hydrochloric acid to obtain the main extract.

[0026] Example 2 (Test Method and Test Results) Next, the test method and results of the pharmacological test demonstrating the GalNAcT expression promoting effect of the extract obtained in Example 1 above in intervertebral disc cells are shown.

[0027] Cells and reagents In Test Examples 1 to 4, human nucleus pulposus cells were used, which were prepared according to the following procedure with the approval of the Tokai University School of Medicine Experimental Ethics Committee. Nucleus pulposus tissue was collected intraoperatively from five patients with herniated discs (ages 29 to 38), three males and two females, with their consent. The nucleus pulposus tissue was cut into small pieces and treated with TrypLE Express (Gibco) for 1 hour, followed by treatment with 0.25 mg / ml Collagense-P (Roche) for 2 hours. The isolated cells were washed twice with α-MEM medium (Wako Chemical) at 37°C and collected at approximately 5 × 10 3 pieces / cm 2Cells were seeded at a density of 100 μg / ml. Cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U / ml penicillin (Gibco), and 100 mg / ml streptomycin (Gibco) under hypoxic conditions of 2% O and 5% CO at 37°C. The medium was changed twice a week, and cells were treated with trypsin (Gibco) and subcultured before reaching confluence. Cells obtained from the third passage were used for individual experiments.

[0028] 25cm of nucleus pulposus cells 2 5000 cells / cm in a flask 2 The cells were seeded at a density of 100 μg / ml and cultured overnight in α-MEM medium containing 10% FBS before adding the extract. The cells were then treated with α-MEM medium supplemented with the extract, 10% FBS, and 170 μM ascorbic acid. The culture medium was changed every other day for two weeks. Nucleus pulposus cells that reached confluence were used in the following experiments.

[0029] statistical analysis Statistical analysis was performed using repeated measures analysis of variance. When a p value of less than 0.05 was obtained, a Bonferroni post-hoc test was performed.

[0030] Test Example 1: Effect on GAG expression (GAG and DNA analysis) Cultured cells were washed with Dulbecco's phosphate-buffered saline (DPBS, DS-Pharma) and treated overnight at 65°C with a buffer containing 25 mg / ml papain (Sigma-Aldrich), 8 mg / ml sodium acetate (Wako Chemical), 4 mg / ml ethylenediaminetetraacetic acid (Sigma-Aldrich), and 1.57 mg / ml L-cysteine ​​(Sigma-Aldrich). Sulfated GAG content was calculated by measuring absorbance at 656 nm using a SPECTRA MAX i3 spectrophotometer (Molecular Devices) with chondroitin-6-sulfate (Biocolor) as a standard and 1,9-dimethyl-methylene blue (Biocolor). DNA content was calculated using the PicoGreen assay (ThermoFisher Scientific, Waltham, MA) on the same spectrophotometer as above, with excitation at 480 nm and emission at 520 nm. The ratio of GAG to DNA content (mean ± standard error) was calculated. An example of the results of the above test is shown in Table 1.

[0031] [Table 1]

[0032] The GAG / DNA value increased 1.7-fold in the 0.1 mNU / mL administration group and 1.4-fold in the 1.0 mNU / mL administration group compared to the control group, and a significant increase in GAG production was observed in the 0.1 mNU / mL administration group of this extract (Table 1).

[0033] Test Example 2: Effect on expression of CSGALNACT1, ANG1 and IGF genes (quantitative real-time PCR method) One week after addition of 1.0 mNU / mL of this extract, cells were harvested and homogenized in lysis buffer. Total RNA (tRNA) was prepared using the SV Total RNA Isolation System (Promega). For each sample, 2 μg of tRNA was reverse transcribed into cDNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). The amount of GALNACT1 mRNA was calculated using the comparative CT method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH: product name, pre-developed TaqMan Assay Reagents (Applied Biosystems)) as an internal standard. The following primers and probes (Applied Biosystems) were used: IGF1 (TaqMan Assay ID: Hs03986524_m1), ANGPT1 (TaqMan Assay ID: Hs00181613_m1), CSGALNACT1 (TaqMan Assay ID: Hs00218054_m1). The expression level of CSGALNACT1, ANGPT1, or IGF1 in the control was set to 1, and the ratio (mean ± standard error) of each group was calculated. Examples of the results of the above test are shown in Tables 2 to 4.

[0034] [Table 2] [Table 3] [Table 4]

[0035] The expression level of CSGALNACT1 was significantly increased by adding 0.1 mNU / mL and 1.0 mNU / mL of this extract (Table 2). Furthermore, the expression levels of ANGPT1 and IGF1 mRNA were significantly increased by adding 0.1 mNU / mL and 1.0 mNU / mL of this extract (Tables 3 and 4).

[0036] Test Example 3: Effect on mRNA expression of CSGALNACT1 and CSGALNACT2 (microarray method) Gene expression in patient-derived nucleus pulposus cells treated with 1.0 mNU / mL of this extract and untreated cells was compared using a microarray. The extract and 170 μM ascorbic acid were added. tRNA was prepared as in Test Example 2, and Cy3-labeled cRNA was prepared using a Low Input Quick Amp Labeling Kit (Agilent Technology). The resulting Cy3-labeled cRNA was hybridized using a SurePrint G3 Human GE 8x60K v2 Microarray (Agilent Technology) and a Gene Expression Hybridization Kit (Agilent Technology). Analysis was then performed using an Agilent DNA microarray scanner (Agilent Technology, G2600D SG13164306) according to the Agilent G3_HiSen_GX_1Color (Agilent Technology) protocol.

[0037] The fluorescence intensity of each probe was converted to an expression value using Agilent Feature Extraction 11.5.1.1 (Agilent Technology). Gene expression analysis software, Gene Spring ver. 13 (Agilent Technology), was used to detect highly expressed genes. Genes involved in GAG synthesis were selected using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2017 Tool and the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database. The expression level after addition of the extract was calculated as a ratio to the expression level after the same incubation period (control) without the extract. Signals from multiple probes on the array for the same gene were averaged and used as a single data set. An example of the results of the above test is shown in Table 5.

[0038] [Table 5]

[0039] The addition of 1.0 mNU / mL of this extract increased the expression of many of the genes involved in GAG synthesis (Table 5). For CSGALNACT, the expression of CSGALNACT1 was increased 1.54-fold and that of CSGALNACT2 was increased 1.12-fold (Table 5).

[0040] Test Example 4: Effect of CSGALNACT1 on protein expression (Western blotting) Protein expression in nucleus pulposus cells treated with this extract and untreated cells was compared using Western blotting. Nucleus pulposus cells were plated in a 6-well plate at 5000 cells / cm. 2 The cells were seeded at a density of 1000 μg / mL, and the extract (0.1 or 1.0 mNU / mL) and 50 μg / mL ascorbic acid diphosphate (AsAP) were added every other day, and the cells were harvested at 1 and 2 weeks later. The cells were lysed in ice-cold cell lysis buffer (50 mM Tris-HCl (pH 7.5), Wako Pure Chemical, 1% Triton X-100, Wako Pure Chemical, and 2 mM CaCl, Sigma-Aldrich) containing protease and phosphatase inhibitors (0.5 mM phenylmethylsulfonyl fluoride, Sigma-Aldrich; 1 / 50 Complete protease inhibitor cocktail, Roche Molecular Biochemical; 1 mM NaVO, Sigma-Aldrich, and 1 mM NaF, Sigma-Aldrich).

[0041] Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (3 μg) were separated and specifically detected by immunoblotting using the following antibodies: anti-CSGANACT1 rabbit polyclonal antibody (Ab83071, Abcam); as a loading control, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (G9545, Sigma-Aldrich). The antibody concentrations were 1:500 for CSGALNACT1 and 1:2000 for GAPDH.

[0042] Equal amounts of protein were diluted with SDS sample buffer and boiled for 5 minutes. Then, electrophoresis was performed on an SDS-polyacrylamide gel. Protein bands separated by electrophoresis were transferred from the gel to a polyvinylidene difluoride membrane (PVDF, BioRad). After washing, the membrane was blocked with 3% bovine serum albumin (BSA, Serologicals) in Tris-buffered saline (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween-20) for 1 hour at room temperature. The primary antibodies listed above were applied to the membrane in 1% BSA / TBST and incubated overnight at 4°C. After further rinsing with TBST, the PVDF membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (GE Healthcare) for 1 hour at room temperature. The bands were imaged using chemiluminescence (ECL Plus, GE Healthcare). The ratio of each group to the GAPDH expression level was calculated. An example of the results of the above test is shown in Figure 1 and Table 6.

[0043] Addition of 0.1 mNU / mL of this extract resulted in enhanced expression of CSGANACT1 protein (Figure 1 and Table 6).

[0044] [Table 6]

[0045] In view of the above, preferred embodiments of the present invention include the following, but are not limited to these.

[0046] (1) An N-acetylgalactosaminyltransferase expression promoter containing an extract from inflamed tissue inoculated with vaccinia virus. (2) The expression promoter described in (1), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1. (3) The expression promoter described in (1), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2. (4) An expression promoter according to any one of (1) to (3), characterized in that the expression promoting effect of N-acetylgalactosaminyltransferase 1 is stronger than the expression promoting effect of N-acetylgalactosaminyltransferase 2. (5) The expression promoter according to any one of (1) to (4), wherein the inflamed tissue is inflamed skin tissue of a rabbit. (6) The expression promoter according to any one of (1) to (5), which is an injection. (7) The expression promoter according to any one of (1) to (5), which is an oral agent.

[0047] (8) A method for determining or evaluating an extract from inflamed tissue inoculated with vaccinia virus or a preparation containing the same, using the activity of promoting the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells as an indicator. (9) The method for determining or evaluating according to (8), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1. (10) The method for determining or evaluating according to (8), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2. (11) A method for determining or evaluating described in any one of (8) to (10), in which the expression increase rates of N-acetylgalactosaminyltransferase 1 and N-acetylgalactosaminyltransferase 2 are compared and it is confirmed that the expression increase rate of N-acetylgalactosaminyltransferase 1 is greater. (12) The method for determining or evaluating the inflamed tissue according to any one of (8) to (11), wherein the inflamed tissue is inflamed skin tissue of a rabbit.

[0048] (13) A method for ensuring the quality standard of an extract from inflamed tissue inoculated with vaccinia virus or a preparation containing the extract by carrying out the judgment or evaluation described in any one of (8) to (12) above. (14) A method for ensuring the quality standard described in (13) above, wherein the formulation is an injection or oral formulation.

[0049] (15) Use of an extract from inflamed tissue inoculated with vaccinia virus for producing an N-acetylgalactosaminyltransferase expression promoter. (16) The use according to (15), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1. (17) The use according to (15), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2. (18) The use according to any one of (15) to (17), characterized in that the N-acetylgalactosaminyltransferase expression promoter has a stronger effect of promoting the expression of N-acetylgalactosaminyltransferase 1 than of N-acetylgalactosaminyltransferase 2. (19) The use according to any one of (15) to (18), wherein the inflamed tissue is inflamed skin tissue of a rabbit. (20) The use according to any one of (15) to (19), wherein the N-acetylgalactosaminyltransferase expression promoter is an injectable preparation. (21) The use according to any one of (15) to (19), wherein the N-acetylgalactosaminyltransferase expression promoter is an oral agent. [Industrial Applicability]

[0050] As described above, the present extract has the effect of promoting the expression of N-acetylgalactosaminyltransferase, in particular, promoting the expression of N-acetylgalactosaminyltransferase 1 more strongly than that of N-acetylgalactosaminyltransferase 2. Therefore, an N-acetylgalactosaminyltransferase expression promoter containing the present extract is useful as a therapeutic or preventive agent for diseases associated with intervertebral disc degeneration and OA.

Claims

1. A method for assuring the quality standards of an extract from inflamed tissue inoculated with vaccinia virus or a pharmaceutical composition containing the extract as an active ingredient, by testing the promoting effect of N-acetylgalactosaminyltransferase and angiopoietin-1 expression in intervertebral disc cells for each production lot of the extract or pharmaceutical composition, and using as an indicator whether both are significantly enhanced compared to a control.

2. The method according to claim 1, wherein the pharmaceutical composition is an injectable or oral preparation.

3. 3. The method according to claim 1, wherein the indicator is one of indicators of the therapeutic effect of an extract from inflamed tissue inoculated with vaccinia virus or a pharmaceutical composition containing the extract as an active ingredient on degenerative disc disease and / or osteoarthritis.

4. The method according to claim 3, wherein the degenerative disc disease is lower back pain.

5. The method according to any one of claims 1 to 4, wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1.

6. The method according to any one of claims 1 to 4, wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2.

7. The method according to any one of claims 1 to 6, wherein in the test for N-acetylgalactosaminyltransferase, the expression-promoting effects of N-acetylgalactosaminyltransferase 1 and N-acetylgalactosaminyltransferase 2 are compared with those of a control, and whether the expression-promoting effect of N-acetylgalactosaminyltransferase 1 is significantly enhanced is used as an indicator.

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