N-acetylgalactosamine transferase expression promoter containing vaccinia virus inoculated inflammatory tissue extract

Abstract

To provide, e.g., an agent for promoting expression of N-acetylgalactosaminyltransferase that contains an extract from inflamed tissues inoculated with the vaccinia virus.SOLUTION: The present invention demonstrated that the extract from inflamed tissues inoculated with the vaccinia virus promotes the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells. Thus, the extract from inflamed tissues inoculated with the vaccinia virus or a preparation containing the extract is useful, e.g., 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 pharmaceutical uses of vaccinia virus-infected inflammatory tissue extract (hereinafter sometimes referred to as "this extract"). More specifically, it relates to an N-acetylgalactosamine transferase (GalNAcT) expression promoter containing this extract, etc.

Background Art

[0002] The intervertebral disc is composed of three tissues with different biochemical and physical properties: the nucleus pulposus, annulus fibrosus, and cartilage endplate. Abundant proteoglycans in the nucleus pulposus retain a large amount of water, giving the intervertebral disc the characteristic that about 80% of it is water. Intervertebral disc degeneration is one of the main causes of low back pain and leads to the development of diseases such as intervertebral disc herniation and spinal canal stenosis. Although there are large individual differences in intervertebral disc degeneration associated with aging, changes in constituent cells in the nucleus pulposus, subsequent matrix changes, and subsequent breakdown of the annulus fibrosus structure are considered to be strongly related to intervertebral disc degeneration. After the age of 60 in humans, the proteoglycan content in the intervertebral disc decreases from 65% in the 10s to about 30% or less, which is half. As a result, the water content in the intervertebral disc decreases. Along with this, it becomes easier to cause structural breakdown due to external factors.

[0003] Proteoglycan is a compound in which polysaccharides (glycosaminoglycans, GAGs) consisting of disaccharide units such as chondroitin sulfate are bound to multiple sites in a tetrasaccharide binding region where a carbohydrate is bound to a serine residue of a core protein. Proteoglycan, which has various functionalities as a biological component, exists in the extracellular matrix and cell surfaces in various major organs, the brain, skin, and tissues throughout the body, and also exists as a main component of cartilage.

[0004] GAGs are classified into four types based on differences in their basic disaccharide structure: (1) heparin and heparan sulfate, (2) chondroitin sulfate and dermatan sulfate, (3) keratan sulfate, and (4) hyaluronic acid. These basic structures undergo various modifications, mainly sulfation. In most cases, GAGs exist bound to a core protein, i.e., as proteoglycans. Various glycosyltransferases and sulfotransferases are known to be involved in the biosynthesis of GAGs.

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

[0006] Chondroitin sulfate is a linear sulfated glycan formed by the alternating polymerization of N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcUA) disaccharides. Two types of N-acetylgalactosamine transferases are involved in the biosynthesis of the chondroitin sulfate glycan backbone. One is N-acetylgalactosamine transferase 1 (GalNAcT1), which transfers GalNAc to the GlcUA residue at the end of the binding region, and the other is N-acetylgalactosamine transferase 2 (GalNAcT2), which transfers GalNAc to the GlcUA terminus of the disaccharide repeat structure. The biosynthesis of chondroitin sulfate begins with the transfer of GalNAc to the GlcUA residue at the end of the binding region by GalNAcT1. Subsequently, GlcUA is transferred by glucuronosyltransferase, and then GalNAc is transferred by GalNAcT2, alternatingly, synthesizing the disaccharide repeat region and extending the glycan. Finally, the constituent sugars are sulfated by sulfotransferase to form chondroitin sulfate. Thus, it has become clear that in the biosynthesis of chondroitin sulfate, GalNAcT1 is involved in the initiation of sugar chain synthesis, and GalNAcT2 is involved in sugar chain elongation. In other words, GalNAcT1 is involved in increasing the number of sugar chains in chondroitin sulfate, rather than in the elongation of the sugar chains.

[0007] The vaccinia virus-inoculated inflammatory tissue extract (this extract) contained in the N-acetylgalactosamine transferase expression promoter according to the present invention, or preparations containing this extract, have analgesic, sedative, anti-stress, anti-allergic, immunostimulatory, anticancer, cirrhosis-suppressing, therapeutic effects against idiopathic thrombocytopenic purpura, therapeutic effects against postherpetic neuralgia, cerebral edema, dementia, spinocerebellar degeneration, etc., therapeutic effects against Raynaud's syndrome, diabetic neuropathy, sequelae of subacute myelopathy-mediated neuropathy, etc., kallikrein production inhibition, peripheral circulatory disorder improvement, bone atrophy improvement, and sepsis and endotoxin It is known to have a wide range of effects, including inhibiting nitric oxide production, which is effective in treating shock; therapeutic effects against osteoporosis; therapeutic effects against AIDS based on inhibitory effects on Nef action and chemokine production; therapeutic effects against ischemic diseases such as cerebral infarction; therapeutic effects against fibromyalgia; therapeutic effects against infectious diseases; preventive or mitigating effects on peripheral neuropathy caused by anticancer drugs; therapeutic effects on chronic prostatitis, interstitial cystitis and / or urinary disorders; promoting the production of neurotrophic factors such as BDNF; liver protection; promoting the migration of pluripotent stem cells (Muse cells); and preventive or therapeutic effects on muscle damage. In addition to these, this extract or preparations containing it are known to promote the synthesis of collagen and proteoglycans in chondrocytes (see Patent Document 1). However, it has not been previously known that this extract or preparations containing it promote the expression of GalNAcT, or that it promotes the expression of GalNAcT1 more strongly than GalNAcT2. The fact that it promotes GalNAcT1 expression more strongly than GalNAcT2 indicates that its effect of increasing the number of glycans is stronger than that of chondroitin sulfate's effect of elongating glycans. 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 [Overview of the project] [Problems that the invention aims to solve]

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

[0010] The inventors of this invention conducted intensive research on the pharmacological effects of this extract and, as a result, discovered that this extract has excellent GalNAcT expression-promoting properties, thus completing the present invention. [Effects of the Invention]

[0011] Because this extract promotes GalNAcT expression, preparations containing this extract can be excellent therapeutic or preventive agents for diseases associated with intervertebral disc degeneration and osteoarthritis. In particular, preparations containing this extract have been used for many years as safe drugs with few side effects, making this invention extremely useful. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is an electrophoresis diagram showing the results of examining the expression level of CSGALNACT1 protein in nucleus pulposus cells treated with this extract, using Western blotting. [Modes for carrying out the invention]

[0013] This extract contains a non-protein active substance extracted and isolated from the inflamed tissue of animals that have been inoculated with vaccinia virus and developed smallpox. While the extract is liquid in its extracted state, it can be solidified by drying. This preparation is highly useful as a pharmaceutical. A specific product manufactured and sold by the applicant in Japan as part of this preparation is "Preparation Containing Vaccinia Virus-Inoculated Rabbit Inflammatory Skin Extract" (product name: Neurotropin / NEUROTROPIN®) (hereinafter referred to as "Neurotropin"). Neurotropin is available as both an injection and a tablet, both of which are medical drugs (ethical drugs).

[0014] The indications for Neurotropin injection are "low back pain, cervicobrachial syndrome, symptomatic neuralgia, itching associated with skin diseases (eczema, dermatitis, urticaria), allergic rhinitis, and coldness, abnormal sensation, and pain as sequelae of subacute myelopathy-mediated neuropathy (SMON)." The indications for Neurotropin tablets are "postherpetic neuralgia, low back pain, cervicobrachial syndrome, periarthritis of the shoulder joint, and osteoarthritis." This preparation was created and developed by the applicant as a pharmaceutical product, and its excellent features in efficacy and safety have been recognized, leading to its long-term sales and establishment of a solid position in the Japanese pharmaceutical market.

[0015] The vaccinia virus-inoculated inflammatory tissue extract in the present invention can be obtained by crushing inflammatory tissue that has been inoculated with vaccinia virus and developed pox, removing tissue fragments by adding an extraction solvent, performing a deproteinization treatment, adsorbing the tissue onto an adsorbent, and then eluting the active ingredient. That is, for example, the process is as follows. (A) Skin tissue, etc., from rabbits, mice, etc., that have been inoculated with vaccinia virus and developed pox is collected, the developed tissue is crushed, and an extraction solvent such as water, phenol water, physiological saline, or phenol-added glycerin water is added, and then the extract (filtrate or supernatant) is obtained by filtration or centrifugation. (B) The extract is heated to an acidic pH and deproteinized. The deproteinized solution is then heated to an alkaline pH and filtered or centrifuged. (C) The obtained filtrate or supernatant is made acidic and adsorbed onto an adsorbent such as activated carbon or kaolin. (D) By adding an extraction solvent such as water to the adsorbent and adjusting the pH to an alkaline level, a vaccinia virus inoculated inflammatory tissue extract can be obtained by eluting the adsorbed components. Subsequently, if desired, the eluate can be evaporated to dryness or freeze-dried under reduced pressure to obtain a dry product.

[0016] For obtaining inflammatory tissue by inoculating with vaccinia virus, various animals that can be infected with vaccinia virus can be used, such as rabbits, cattle, horses, sheep, goats, monkeys, rats, and mice. Rabbit inflammatory skin tissue is preferred as the inflammatory tissue. Any rabbit belonging to the order Lagomorpha is acceptable. Examples include European rabbits, domestic rabbits (domesticated European rabbits), Japanese hares (Hare), pikas, and snowshoe hares. Of these, domestic rabbits are suitable for use. In Japan, there is a breed called "ie-usagi" (domestic rabbit) that has been bred for a long time and widely used as livestock or laboratory animals, which is also another name for domestic rabbits. There are many breeds of domestic rabbits, but breeds such as the Japanese White and New Zealand White (New Zealand White) can be suitably used.

[0017] Any strain of vaccinia virus may be used. Examples include the Lister strain, Dairen strain, Ikeda strain, EM-63 strain, and the New York City Board of Health strain.

[0018] The basic extraction steps (A) to (D) of this extract described above can be carried out in more detail, for example, as follows. Regarding process (A) Inflamed skin tissue is collected by intradermal inoculation of vaccinia virus into the skin of a rabbit to cause pox. The collected skin tissue is washed and disinfected with a phenol solution or the like. This inflamed skin tissue is crushed, and an extraction solvent 1 to 5 times the amount thereof is added. Here, "crushing" means finely crushing into minced form using a mincing machine or the like. As the extraction solvent, distilled water, physiological saline, a buffer solution of weak acidity to weak basicity, etc. can be used, and bactericides and preservatives such as phenol, stabilizers such as glycerin, salts such as sodium chloride, potassium chloride, magnesium chloride, etc. may be appropriately added. At this time, cell tissue can also be destroyed by treatments such as freeze-thawing, ultrasonic waves, cell membrane-lysing enzymes or surfactants to facilitate extraction. The obtained suspension is left to stand for 5 to 12 days. During that time, it may be heated to 30 to 45 °C with or without appropriate stirring. The obtained liquid is subjected to solid-liquid separation (such as filtration or centrifugation) to remove tissue pieces and obtain a crude extract (filtrate or supernatant).

[0019] Regarding step (B) The crude extract obtained in step (A) is subjected to a protein removal treatment. Protein removal can be carried out by known methods commonly used, and methods such as heat treatment, treatment with a protein denaturing agent (for example, organic solvents such as acids, bases, urea, guanidine, acetone, etc.), isoelectric point precipitation, salting out, etc. can be applied. Next, a normal method for removing insolubles, for example, filtration using filter paper (such as cellulose, nitrocellulose, etc.), glass filter, celite, Zietz filter plate, etc., ultrafiltration, centrifugation, etc. is used to obtain a filtrate or supernatant from which precipitated insoluble proteins have been removed.

[0020] Regarding step (C) The filtrate or supernatant obtained in step (B) is adjusted to acidic, preferably pH 3.5 to 5.5, and an adsorption operation to an adsorbent is carried out. Adsorbents that can be used include activated carbon, kaolin, etc. The adsorbent can be added to the extract and stirred, or the extract can be passed through an adsorbent-packed column to adsorb the active ingredient to the adsorbent. When the adsorbent is added to the extract, the solution can be removed by filtration, centrifugation, etc. to obtain an adsorbent adsorbed with the active ingredient.

[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 9 to 12, eluted at room temperature or by heating or stirring as appropriate, and the adsorbent is removed by conventional methods such as filtration or centrifugation. As the elution solvent, a basic solvent such as water, methanol, ethanol, isopropanol, etc., adjusted to a basic pH, or a suitable mixture thereof can be used, and water adjusted to a pH of 9 to 12 can be used. The amount of elution solvent can be set as appropriate. The eluate thus obtained can be used as a drug ingredient, and the pH can be adjusted to near neutral as appropriate to finally obtain vaccinia virus-inoculated rabbit inflammatory skin extract (this extract).

[0022] Since this extract is liquid when prepared, it can be concentrated or diluted to the desired concentration. When manufacturing a formulation from this extract, heat sterilization is preferable. To prepare an injectable formulation, for example, sodium chloride can be added to prepare an isotonic solution with physiological saline. It can also be administered orally in liquid or gel form, but by performing appropriate concentration and drying operations on this extract, oral solid formulations such as tablets can also be manufactured. Specific methods for manufacturing such oral solid formulations from this extract are described in the specifications of Japanese Patent No. 3818657 and No. 4883798. The injectable and oral formulations obtained in this manner are examples of this formulation.

[0023] The following describes examples of methods for producing this extract, as well as the results of pharmacological tests concerning the novel pharmacological effects of this extract and its ability to promote the expression of N-acetylgalactosamine transferase. However, the present invention is not limited in any way by the descriptions of these examples. [Examples]

[0024] Example 1: Preparation of the extract Vaccinia virus was intradermally inoculated into the skin of healthy, mature rabbits, and the infected skin was excised and collected. The collected skin was washed and disinfected with phenol solution, excess phenol solution was removed, the skin was crushed, phenol solution was added and mixed, and left for 3 to 7 days. Then, it was heated at 35 to 40°C with stirring for another 3 to 4 days. After that, the extract obtained by solid-liquid separation was adjusted to pH 4.5 to 5.2 with hydrochloric acid, heated at 90 to 100°C for 30 minutes, filtered, and deproteinized. Furthermore, the filtrate was adjusted to pH 9.0 to 9.5 with sodium hydroxide, heated at 90 to 100°C for 15 minutes, and then solid-liquid separation was performed.

[0025] The deproteinized solution was adjusted to pH 4.0-4.3 with hydrochloric acid, and 2% of the volume of activated carbon was added. After stirring for 2 hours, solid-liquid separation was performed. 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, and then centrifuged to obtain the supernatant. Water was again added to the precipitated activated carbon, and the pH was adjusted to 10.5-11 with sodium hydroxide. The mixture was stirred at 60°C for 90-100 minutes, and then centrifuged to obtain the supernatant. Both supernatants were combined and neutralized with hydrochloric acid to obtain the extract.

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

[0027] Cells and reagents In Experimental Examples 1 to 4, human nucleus pulposus cells prepared according to the following procedure were used with the approval of the Experimental Ethics Committee of Tokai University School of Medicine. Nucleus pulposus tissue was intraoperatively collected from a total of five patients with herniated discs (ages 29 to 38 years) with their consent. The nucleus pulposus tissue was cut into small pieces, treated with TrypLE Express (Gibco) for 1 hour, and then treated with 0.25 mg / ml Collagenense-P (Roche) for 2 hours. The isolated cells were washed twice with α-MEM medium (Wako Chemical) at 37°C and then subjected to approximately 5 × 10⁴ 3 pieces / cm 2Cells were seeded at a density of [density]. Cells were cultured at 37°C under hypoxic conditions of 2% O2 and 5% CO2 in α-MEM medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U / ml penicillin (Gibco), and 100 mg / ml streptomycin (Gibco). The medium was changed twice a week, and cells were treated with trypsin (Gibco) before reaching confluence and then subcultured. Cells obtained from the 3rd subgeneration were used for individual experiments.

[0028] Nucleus pulposus cells 25 cm 2 5000 pieces / cm in a flask 2 The cells were seeded at a density and cultured overnight in α-MEM medium containing 10% FBS before adding the extract. Subsequently, the cells were treated with α-MEM medium supplemented with the extract, 10% FBS, and 170 μM ascorbic acid. The culture was maintained for two weeks, with the medium changed every other day. Confluent nucleus pulposus cells were used in the following tests.

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

[0030] Test Example 1: Effects 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 using a SPECTRA MAX i3 (Molecular Devices) spectrophotometer, measuring the absorbance at 656 nm with 1,9-dimethyl methylene blue (Biocolor) as the standard, using chondroitin-6-sulfate (Biocolor). DNA content was calculated using the same spectrophotometer by the PicoGreen assay (ThermoFisher Scientific Waltham, MA), with excitation at 480 nm and emission at 520 nm. The ratio of GAG to DNA content (mean ± standard error) was calculated. Table 1 shows an example of the results of the above test.

[0031] [Table 1]

[0032] GAG / DNA levels increased 1.7 times in the 0.1 mNU / mL dose group and 1.4 times in the 1.0 mNU / mL dose group compared to the control group, indicating a significant enhancement of GAG production in the 0.1 mNU / mL dose group of this extract (Table 1).

[0033] Test Example 2: Effects on the expression of CSGALNACT1, ANG1, and IGF genes (quantitative real-time PCR method) One week after adding 1.0 mNU / mL of the extract, cells were collected, homogenized in lysis buffer, and 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 by comparative CT using 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), and CSGALNACT1 (TaqMan Assay ID: Hs00218054_m1). The ratio (mean ± standard error) of each group was calculated when the expression level of CSGALNACT1, ANGPT1, or IGF1 in the control group was set to 1. An example of the results of the above study is shown in Tables 2 to 4.

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

[0035] The expression levels of CSGALNACT1 were significantly enhanced by the addition of this extract at concentrations of 0.1 mNU / mL and 1.0 mNU / mL (Table 2). Furthermore, the mRNA expression levels of ANGPT1 and IGF1 were also significantly enhanced by the addition of this extract at concentrations of 0.1 mNU / mL and 1.0 mNU / mL (Tables 3 and 4).

[0036] Test Example 3: Effects of CSGALNACT1 and CSGALNACT2 on mRNA expression (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 was added to 170 μM ascorbic acid. tRNA was prepared in the same manner as in Test Example 2, and Cy3-labeled cRNA was prepared using the Low Input Quick Amp Labeling Kit (Agilent Technology). The resulting Cy3-labeled cRNA was hybridized using the SurePrint G3 Human GE 8x60K v2 Microarray (Agilent Technology) and the Gene Expression Hybridization Kit (Agilent Technology). Subsequently, the DNA was analyzed 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 expression values ​​using the Agilent Feature Extraction 11.5.1.1 (Agilent Technology) digital conversion software. Genes with high expression levels were detected using the Gene Spring ver.13 (Agilent Technology) gene expression analysis software. 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 ratio of expression levels after addition was calculated, with the expression level after addition set to 1 when cultured for the same time without the extract (control). The signals from multiple probes on the array for the same gene were averaged and used as a single data point. An example of the results of the above test is shown in Table 5.

[0038] [Table 5]

[0039] Many genes involved in GAG synthesis showed enhanced expression upon addition of 1.0 mNU / mL of this extract (Table 5). In CSGALNACT, CSGALNACT1 showed a 1.54-fold increase in expression, and CSGALNACT2 showed a 1.12-fold increase (Table 5).

[0040] Test Example 4: Effect of CSGALNACT1 on protein expression (Western blot method) Protein expression in nucleus pulposus cells treated with this extract and those that were not treated was compared using Western blotting. 5000 nucleus pulposus cells / cm³ were placed in a 6-well plate. 2 Seeds were seeded at a density of [density], and from the following day, the extract (0.1 or 1.0 mNU / mL) and 50 μg / mL ascorbic acid diphosphate (AsAP) were added every other day. Cells were harvested at 1 and 2 weeks later. The cells were lysed in a cell lysis buffer (50 mM Tris-HCl (pH 7.5), Wako Pure Chemical, 1% Triton X-100, Wako Pure Chemical, and 2 mM CaCl2, Sigma-Aldrich) containing ice-cooled protease inhibitors and phosphatase inhibitors (0.5 mM phenylmethylsulfonyl fluoride, Sigma-Aldrich; 1 / 50 Complete protease inhibitor cocktail, Roche Molecular Biochemical; 1 mM Na3VO4, Sigma-Aldrich; and 1 mM NaF, Sigma-Aldrich).

[0041] Protein concentration was measured using the BCA Protein Analysis Kit (Thermo Fisher Science), and an equal amount of protein (3 μg) was isolated and specifically detected by immunoblotting using the following antibodies. The antibodies used were anti-CSGANACT1 rabbit polyclonal antibody (Ab83071, Abcam) and, 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 volumes of protein were diluted in SDS sample buffer and boiled for 5 minutes. Electrophoresis was then performed using an SDS-polyacrylamide gel. The separated protein bands 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 (50mM Tris (pH 7.6), 150mM NaCl, 0.1% Tween-20) at room temperature for 1 hour. The primary antibody dissolved in 1% BSA / TBST was applied to this membrane and incubated overnight at 4°C. The PVDF membrane was then washed with TBST and reacted with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (GE Healthcare) at room temperature for 1 hour. The bands were imaged using chemifluorescence (ECL Plus, GE Healthcare). The ratio of each group was calculated relative to the GAPDH expression level (set to 1). An example of the results of the above test is shown in Figure 1 and Table 6.

[0043] The addition of this extract at a concentration of 0.1 mNU / mL enhanced the expression of the CSGANACT1 protein (Figure 1 and Table 6).

[0044] [Table 6]

[0045] Based on the above, preferred embodiments of the present invention include, but are not limited to, the following.

[0046] (1) An N-acetylgalactosamine transferase expression promoter containing vaccinia virus inoculated inflammatory tissue extract. (2) The expression promoter described in (1), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1. (3) The expression promoter described in (1), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2. (4) An expression promoter according to any one of (1) to (3), characterized in that the expression-promoting effect of N-acetylgalactosamine transferase 1 is stronger than the expression-promoting effect of N-acetylgalactosamine transferase 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) An injectable expression promoter according to any one of (1) to (5). (7) An oral expression promoter according to any one of (1) to (5).

[0047] (8) A method for determining or evaluating vaccinia virus-inoculated inflammatory tissue extract or a preparation containing the same, using the expression-promoting effect of N-acetylgalactosamine transferase in intervertebral disc cells as an indicator. (9) The determination or evaluation method described in (8), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1. (10) The determination or evaluation method described in (8), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2. (11) A determination or evaluation method according to any one of (8) to (10), which involves comparing the expression increase rates of N-acetylgalactosamine transferase 1 and N-acetylgalactosamine transferase 2 and confirming that the expression increase rate of N-acetylgalactosamine transferase 1 is greater. (12) The determination or evaluation method described in any of (8) to (11) that the inflamed tissue is inflamed skin tissue of a rabbit.

[0048] (13) A method for ensuring the quality standards of vaccinia virus inoculated inflammatory tissue extract or preparations containing the same by performing any of the determinations or evaluations described in (8) to (12) above. (14) A method to ensure that the preparation is an injectable or oral preparation and meets the quality standards described in (13) above.

[0049] (15) Use of vaccinia virus inoculated inflammatory tissue extracts for the production of N-acetylgalactosamine transferase expression promoters. (16) Use as described in (15), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1. (17) Use as described in (15), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2. (18) The use according to any one of (15) to (17), characterized in that the N-acetylgalactosamine transferase expression promoter has a stronger effect in promoting the expression of N-acetylgalactosamine transferase 1 than in promoting the expression of N-acetylgalactosamine transferase 2. (19) Use according to any one of (15) to (18), wherein the inflamed tissue is inflamed skin tissue of a rabbit. (20) Use according to any one of (15) to (19), wherein the N-acetylgalactosamine transferase expression promoter is an injectable preparation. (21) Use according to any one of (15) to (19), wherein the N-acetylgalactosamine transferase expression promoter is an oral agent. [Industrial applicability]

[0050] As described above, this extract has an effect of promoting the expression of N-acetylgalactosamine transferase, and in particular, it promotes the expression of N-acetylgalactosamine transferase 1 more strongly than N-acetylgalactosamine transferase 2. For this reason, an N-acetylgalactosamine transferase expression promoter containing this extract is useful as a treatment or preventive agent for diseases associated with intervertebral disc degeneration and osteoarthritis (OA).

Claims

1. A method for determining or evaluating the therapeutic effect of a vaccinia virus-inoculated inflammatory tissue extract or a pharmaceutical composition containing the same as an active ingredient, wherein for each manufacturing lot, the expression-promoting effect of N-acetylgalactosamine transferase and angiopoietin-1 in intervertebral disc cells is tested, and if both are significantly enhanced compared to a control, the therapeutic effect is determined or evaluated.

2. The determination or evaluation method according to claim 1, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.

3. The determination or evaluation method according to claim 1, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.

4. The method for determining or evaluating a therapeutic effect according to claim 1, wherein, in a test of N-acetylgalactosamine transferase, the expression-promoting effects of N-acetylgalactosamine transferase 1 and N-acetylgalactosamine transferase 2 are compared with a control, and the therapeutic effect is determined or evaluated if the expression-promoting effect of N-acetylgalactosamine transferase 1 is significantly enhanced.

5. The determination or evaluation method according to any one of claims 1 to 4, wherein the inflamed tissue is inflamed skin tissue of a rabbit.

6. A method for ensuring the quality standards of a vaccinia virus-inoculated inflammatory tissue extract or a pharmaceutical composition containing the same as an active ingredient, wherein, in the determination or evaluation method according to any one of claims 1 to 5, the quality standards are determined to be ensured when the expression-promoting effects of N-acetylgalactosamine transferase and angiopoietin-1 are significantly enhanced compared to the control.

7. A method for ensuring the quality standards described in claim 6, wherein the pharmaceutical composition is an injectable or oral preparation.

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