JP2019142853A - Agent for promoting expression of n-acetylgalactosaminyltransferase containing extract from inflamed tissues inoculated with vaccinia virus - Google Patents

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

  The present invention relates to a novel pharmaceutical use and the like of an extract of inflamed tissue inoculated with vaccinia virus (hereinafter sometimes referred to as “the present extract”). More specifically, the present invention relates to an N-acetylgalactosamine transferase (GalNAcT) expression promoter containing this extract.

  The intervertebral disc is composed of three biochemically and physically different tissues: nucleus pulposus, annulus fibrosus, and cartilage endplate, and abundant proteoglycans in the nucleus pulposus retain a lot of water, About 80% of the intervertebral disc is characterized by moisture. Intervertebral disc degeneration is one of the main causes of low back pain and leads to the onset of disc herniation and spinal stenosis. Intervertebral disc degeneration associated with aging varies widely among individuals, but changes in the constituent cells in the nucleus pulposus and the resulting substrate changes, and subsequent disruption of the annulus fibrosis are thought to be strongly related to disc degeneration. . In humans after the 60s, the proteoglycan content in the intervertebral disc decreases from 65% in the teens to about 30%, less than half, resulting in a decrease in the water content of the intervertebral disc. Along with this, structural failure due to external factors is likely to occur.

  A proteoglycan is a compound in which a plurality of polysaccharides (glycosaminoglycans, GAGs) continuous with disaccharide units such as chondroitin sulfate are bound to a tetrasaccharide-binding region in which a carbohydrate is bound to a serine residue of a core protein. Proteoglycans having various functionalities as biological components are present on the extracellular matrix and cell surface in major organs, brain, skin, and whole body tissues, and also exist as the main component of cartilage.

  GAGs are classified into four types: (1) heparin, heparan sulfate, (2) chondroitin sulfate, dermatan sulfate, (3) keratan sulfate, and (4) hyaluronic acid, depending on the basic structure of the disaccharide. These basic skeletons undergo various modifications mainly for sulfation. In many cases, GAG exists in a form bound to the core protein, that is, as a proteoglycan. It is known that various glycosyltransferases and sulfate group transferases are involved in GAG biosynthesis.

  Chondroitin sulfate is a type of GAG and is abundant as a side chain of aggrecan, which is a major component of the extracellular matrix of cartilage. It is known that chondroitin sulfate decreases in cartilage of patients with intervertebral disc degeneration and osteoarthritis (OA).

  Chondroitin sulfate is a linear sulfated sugar chain in which N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcUA) disaccharides are alternately and repeatedly polymerized. Two types of N-acetylgalactosamine transferase are involved in the biosynthesis of the sugar chain skeleton of chondroitin sulfate. One is N-acetylgalactosaminyltransferase 1 (GalNAcT1) that transfers GalNAc to the GlcUA residue at the end of the above binding region, and the other is N-acetylgalactosamine transfer that transfers GalNAc to the GlcUA end of the disaccharide repeat structure. Enzyme 2 (GalNAcT2). The biosynthesis of chondroitin sulfate begins with the transfer of GalNAc by GalNAcT1 to the GlcUA residue at the end of the binding region. Subsequently, GlcUA is transferred alternately by glucuronyltransferase, and then GalNAc is transferred by GalNAcT2, and a repeating region of disaccharide is synthesized to extend the sugar chain. Finally, the constituent sugars are sulfated by sulfotransferase to become chondroitin sulfate. Thus, in the biosynthesis of chondroitin sulfate, it has been clarified that GalNAcT1 is involved in the initiation of sugar chain synthesis and GalNAcT2 is involved in the elongation of sugar chains. In other words, GalNAcT1 is involved in the increase in the number of sugar chains, not the elongation of the chondroitin sulfate sugar chain.

  About vaccinia virus inoculated inflammatory tissue extract (this extract) contained in the N-acetylgalactosamine transferase expression promoter according to the present invention or a preparation containing the same, analgesic action, sedative action, anti-stress action, anti-allergy Action, immunity promoting action, anticancer action, cirrhosis inhibiting action, therapeutic effect for idiopathic thrombocytopenic purpura, postherpetic neuralgia, cerebral edema, dementia, spinocerebellar degeneration, Raynaud's syndrome, diabetic Therapeutic effect on neuropathy, SMON sequelae, etc., Kallikrein production inhibitory action, peripheral circulation disorder improving action, bone atrophy improving action, nitric oxide production suppressing action effective for treatment of sepsis and endotoxin shock, therapeutic effect on osteoporosis, Nef action AIDS treatment effect based on inhibitory action and chemokine production inhibitory action, therapeutic effect on ischemic diseases such as cerebral infarction , Therapeutic effects on fibromyalgia, therapeutic effects on infectious diseases, prevention or alleviation of peripheral neuropathy with anticancer agents, therapeutic effects on chronic prostatitis, interstitial cystitis and / or dysuria, neurotrophic factors such as BDNF A wide variety of actions and effects are known, such as an action of promoting production of hepatitis, an action of protecting liver, an action of promoting migration of pluripotent stem cells (Muse cells), and an effect of preventing or treating muscle damage. In addition to these, the present extract or a preparation containing the extract is known to have an action of promoting the synthesis of collagen and proteoglycan in chondrocytes (see Patent Document 1). However, it has not been known so far that the present extract or a preparation containing the same promotes the expression of GalNAcT, and more strongly promotes the expression of GalNAcT1 than GalNAcT2. Promoting the expression of GalNAcT1 more strongly than GalNAcT2 indicates that the action of increasing the number of sugar chains is stronger than the action of extending the sugar chain of chondroitin sulfate. No drug having such an action has been known so far.

International Publication WO2012 / 051173

  The present invention provides a GalNAcT expression promoter containing this extract.

  As a result of intensive studies on the pharmacological action of the extract, the present inventors have found that the extract has an excellent GalNAcT expression promoting action, and have completed the present invention.

  Since the extract has an action of promoting the expression of GalNAcT, a preparation containing the extract can be an excellent therapeutic or preventive agent for diseases associated with disc degeneration and OA. In particular, since the preparation containing the present extract has been used for many years as a highly safe drug with few problems such as side effects, the present invention is extremely useful.

FIG. 1 is an electrophoretogram showing the results of examining the expression level of CSGALNACT1 protein in nucleus pulposus cells treated with this extract by Western blotting.

  This extract is an extract containing a non-protein active substance extracted and separated from an inflamed tissue of an animal that has been inoculated with vaccinia virus. The extract is liquid in the extracted state, but can also be made solid by drying. This preparation is very useful as a pharmaceutical product. The specific product manufactured and sold by the applicant in Japan as this preparation is “Vaccinia virus-inoculated rabbit inflammation skin extract-containing preparation” (trade name: Neurotropin / registered trademark) (hereinafter “Neurotropin”) .) Neurotropins include injections and tablets, both of which are ethical drugs.

  The indications for injections of neurotropin are “back pain, neck-shoulder syndrome, symptomatic neuralgia, pruritus associated with skin diseases (eczema, dermatitis, urticaria), allergic rhinitis, cold feeling of SMON aftereffects. "Abnormal perception / pain". The indications for neurotropin tablets are “postherpetic neuralgia, low back pain, cervico-arm syndrome, peri-shoulderitis, osteoarthritis”. This formulation was created by the applicant and developed as a pharmaceutical product. Its superior efficacy and safety features have been evaluated, and it has been sold for many years, establishing a firm position in the Japanese pharmaceutical market. Is.

The inflamed tissue extract inoculated with vaccinia virus in the present invention crushes inflamed tissue that has been inoculated with vaccinia virus, and after removing tissue fragments by adding an extraction solvent, the protein is removed and adsorbed by the adsorbent And then eluting the active ingredient. That is, for example, the following steps.
(A) Collect skin tissue such as rabbits and mice inoculated with vaccinia virus, crush the germed tissue, and add an extraction solvent such as water, phenol water, physiological saline or phenol glycerin water. Thereafter, an extract (filtrate or supernatant) is obtained by filtration or centrifugation.
(B) The extract is adjusted to an acidic pH, heated and deproteinized. Next, the deproteinized solution is adjusted to be alkaline and heated, followed by filtration or centrifugation.
(C) The obtained filtrate or supernatant is acidified and adsorbed on an adsorbent such as activated carbon or kaolin.
(D) An extract from inflamed 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 made into a dried product by evaporating to dryness or freeze-drying under reduced pressure.

  As animals for inoculating vaccinia virus to obtain inflamed tissues, various animals infected with vaccinia virus such as rabbits, cows, horses, sheep, goats, monkeys, rats and mice can be used. Inflamed skin tissue is preferred. Any rabbit may be used as long as it belongs to the order of rabbit eyes. Examples include rabbits, rabbits (rabbits made from rabbits), rabbits (Japanese rabbits), rabbits, snow rabbits, and the like. Of these, chira rabbits are suitable for use. In Japan, there is a so-called rabbit that has been bred from the past and used widely as domestic animals or experimental animals. There are many varieties (breeds) of the rabbit, but varieties such as Japanese white varieties and New Zealand white varieties (New Zealand white) can be suitably used.

  The vaccinia virus may be of any strain. Examples include Lister strains, Daren strains, Ikeda strains, EM-63 strains, New York City Board of Health strains, and the like.

More specifically, the basic extraction steps (A) to (D) of the present extract described above can be performed as follows, for example.
Step (A) Inflammated skin tissue obtained by intradermal inoculation of vaccinia virus into the skin of rabbits is collected. The collected skin tissue is washed and disinfected with a phenol solution. This inflamed skin tissue is crushed and 1 to 5 times the amount of extraction solvent is added. Here, crushing means crushing finely into a mince using a mincing machine or the like. In addition, as extraction solvents, distilled water, physiological saline, weakly acidic to weakly basic buffer solutions, and the like can be used. Sterilizers and preservatives such as phenol, stabilizers such as glycerin, sodium chloride, potassium chloride Further, salts such as magnesium chloride may be appropriately added. At this time, extraction can be facilitated by disrupting the cell tissue by treatment with freeze-thaw, ultrasound, cell membrane lytic enzyme, surfactant or the like. The resulting suspension is left for 5 to 12 days. Meanwhile, the mixture may be heated to 30 to 45 ° C. with or without stirring as appropriate. The obtained liquid is subjected to solid-liquid separation (filtration, centrifugation, etc.) to remove the tissue piece to obtain a crude extract (filtrate or supernatant).

About a process (B) A protein removal process is performed about the crude extract obtained at the process (A). Deproteinization can be carried out by known methods commonly used, such as heat treatment, treatment with a protein denaturant (for example, an organic solvent such as acid, base, urea, guanidine, acetone, etc.), isoelectric precipitation, salting out. Etc. can be applied. Subsequently, the insoluble protein that has been precipitated by a usual method for removing insoluble matters, for example, filtration using filter paper (cellulose, nitrocellulose, etc.), glass filter, celite, zeit filtration plate, etc., ultrafiltration, centrifugation, etc. A removed filtrate or supernatant is obtained.

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 the adsorbent is performed. Examples of usable adsorbents include activated carbon, kaolin, and the like. Add the adsorbent to the extract and stir, or pass the extract through an adsorbent-filled column to add the active ingredient to the adsorbent. Can be adsorbed. When an adsorbent is added to the extract, the adsorbent can be obtained by adsorbing the active ingredient by removing the solution by filtration or centrifugation.

About step (D) In order to elute (desorb) the active ingredient from the adsorbent obtained in step (C), an elution solvent is added to the adsorbent and adjusted to basic, preferably pH 9-12, Alternatively, it is eluted by heating or stirring as appropriate, and the adsorbent is removed by a usual method such as filtration or centrifugation. As an elution solvent to be used, a basic solvent, for example, water adjusted to a basic pH, methanol, ethanol, isopropanol or the like, or an appropriate mixed solution thereof can be used, preferably water adjusted to pH 9 to 12. Can be used. The amount of the elution solvent can be appropriately set. In order to use the eluate thus obtained as a drug substance, the pH is appropriately adjusted to near neutral, etc. to finally obtain a vaccinia virus-inoculated rabbit inflammation skin extract (this extract). Can do.

  Since this extract is liquid at the time of completion, it can be made to have a desired concentration by appropriately concentrating and diluting. When manufacturing a formulation from this extract, it is preferable to heat-sterilize. In order to prepare an injection, for example, sodium chloride or the like can be added to prepare a solution that is isotonic with physiological saline. Oral administration in a liquid or gel state is also possible, but an oral solid preparation such as a tablet can be produced by performing an operation such as concentration and drying appropriate for the extract. Specific methods for producing such an oral solid preparation from this extract are described in the specifications of Japanese Patent Nos. 3818657 and 4883798. The injections and oral preparations thus obtained are examples of this preparation.

  Hereinafter, examples of the method for producing the extract, and pharmacological test results regarding the novel pharmacological action of the extract and the expression promoting action of N-acetylgalactosamine transferase will be shown. There are no restrictions.

Example 1 Production of the Extract Vaccinia virus was intradermally inoculated into the skin of a healthy mature rabbit, and the sprouted skin was cut out and collected. The collected skin is washed and disinfected with a phenolic solution, then the excess phenolic solution is removed, crushed, the phenolic solution is added and mixed, left for 3-7 days, and then stirred for an additional 3-4 days Warmed to 35-40 ° C. Thereafter, the extract obtained by solid-liquid separation was adjusted to pH 4.5 to 5.2 with hydrochloric acid, heat-treated at 90 to 100 ° C. for 30 minutes, and then filtered to deproteinize. Further, 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 separated into solid and liquid.

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

Example 2 (Test method and test results)
Next, a test method and test results of a pharmacological test showing the GalNAcT expression promoting action of intervertebral disc cells of the extract obtained in Example 1 above will be shown.

In Cell and Reagent Test Examples 1 to 4, human nucleus pulposus cells that had been approved by the Tokai University School of Medicine Experimental Ethics Committee and were prepared by the following procedure were used. The nucleus pulposus tissue was collected from 5 patients with intervertebral disc herniation (aged 29 to 38 years old) with the consent of three men and two women. The nucleus pulposus tissue was cut into small pieces, treated with TrypLE Express (Gibco) for 1 hour, followed by treatment with 0.25 mg / ml Collagense-P (Roche) for 2 hours. Cells isolated at 37 ° C. were washed twice with α-MEM medium (Wako Chemical) and seeded at a density of about 5 × 10 ³ cells / cm ² . Cells 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 ₂ and The cells were cultured at 37 ° C. under 5% CO ₂ conditions. The medium was changed twice a week and treated with trypsin (Gibco) and subcultured before the cells reached confluence. Cells from passage 3 were used for individual experiments.

Nucleus nucleus cells were seeded at a density of 5000 cells / cm ^{2 in} a 25 cm ² flask and cultured overnight in α-MEM medium containing 10% FBS before addition of the extract. Thereafter, the cells were treated with α-MEM medium supplemented with this extract, 10% FBS and 170 μM ascorbic acid. While maintaining the culture for 2 weeks, the medium was changed every other day. The nucleus pulposus cells that reached confluence were used in the following tests.

Statistical analysis Statistical analysis was repeated measures analysis of variance. When a p-value of less than 0.05 was obtained, Bonferroni post-test was performed.

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

  The GAG / DNA value increased 1.7 times in the 0.1 mNU / mL group and 1.4 times in the 1.0 mNU / mL group compared to the control, and a significant enhancement of GAG production was observed in the 0.1 mNU / mL group of this extract. (Table 1).

Test Example 2 Effect on CSGALNACT1, ANG1 and IGF gene expression (quantitative real-time PCR)
One week after addition of 1.0 mNU / mL of this extract, cells were harvested, 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 mRNA of GALNACT1 was calculated by a comparative CT method 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), CSGALNACT1 (TaqMan Assay ID: Hs00218054_m1). The ratio (average value ± standard error) of each group when the expression level of CSGALNACT1, ANGPT1 or IGF1 in the control was 1 was calculated. Examples of the results of the above tests are shown in Tables 2 to 4.

  The expression level of CSGALNACT1 was found to be significantly enhanced by the addition of 0.1 mNU / mL and 1.0 mNU / mL of this extract (Table 2). In addition, it was found that the mRNA expression levels of ANGPT1 and IGF1 were significantly enhanced by the addition of 0.1 mNU / mL and 1.0 mNU / mL of this extract (Tables 3 and 4).

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

  The fluorescence intensity of each probe was converted into an expression value using numerical conversion software Agilent Feature Extraction 11.5.1.1 (Agilent Technology). Gene expression analysis software Gene Spring ver.13 (Agilent Technology) was used to detect genes with high expression levels. Genes involved in GAG synthesis were selected using Database for Annotation, Visualization and Integrated Discovery (DAVID) 2017 Tool and Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database. The ratio of the expression level after addition when the expression level in the case of culturing for the same time without adding this extract (control) was set to 1 was calculated. Signals from multiple probes on the array for the same gene were averaged and used as one data. An example of the results of the test is shown in Table 5.

  In many of the genes involved in GAG synthesis, expression enhancement was observed when 1.0 mNU / mL of this extract was added (Table 5). In CSGALNACT, CSGALNACT1 increased expression by 1.54 times and CSGALNACT2 increased by 1.12 times (Table 5).

Test Example 4 Effect of CSGALNACT1 on protein expression (Western blotting)
Protein expression in the extract treated and untreated nucleus pulposus cells was compared using Western blotting. Nucleus nucleus cells are seeded in a 6-well plate at a density of 5000 cells / cm ² , and the following day, this extract (0.1 or 1.0 mNU / mL) and 50 μg / mL ascorbic acid diphosphate (AsAP) are added every other day. Cells were collected at 1 and 2 weeks later. The cells were incubated with ice-cold protease and phosphatase inhibitors (0.5 mM phenylmethylsulfonyl fluoride, Sigma-Aldrich, 1/50 Complete protease inhibitor cocktail, Roche Molecular Biochemical, 1 mM Na ₃ VO ₄ , Sigma-Aldrich, and Lysis was performed with a cell lysis buffer (50 mM Tris-HCl (pH 7.5), Wako Pure Chemical, 1% Triton X-100, Wako Pure Chemical and 2 mM CaCl ₂ , Sigma-Aldrich) containing 1 mM NaF, Sigma-Aldrich).

  The protein concentration was measured using a BCA protein analysis kit (Thermo Fisher Science), and an equal amount of protein (3 μg) was separated and specifically detected by immunoblotting using the following antibodies. The antibody used was an anti-CSGANACT1 rabbit polyclonal antibody (Ab83071, Abcam), and an anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (G9545, Sigma-Aldrich) as a loading control. The antibody concentration was 1: 500 for CSGALNACT1 and 1: 2000 for GAPDH.

  An equal amount of protein was diluted with SDS sample buffer and boiled for 5 minutes. Thereafter, electrophoresis was performed using SDS-polyacrylamide gel. The protein bands separated by electrophoresis are transferred from the gel to a polyvinylidene difluoride membrane (PVDF, BioRad), and the membrane is washed and then tris-buffered saline (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween-20) ) With 3% bovine serum albumin (BSA, Serologicals) for 1 hour at room temperature. The above primary antibody dissolved in 1% BSA / TBST was applied to the membrane, and incubated at 4 ° C. overnight. Further, the PVDF membrane was washed with TBST, and then reacted with an anti-rabbit IgG secondary antibody (GE Healthcare) conjugated with horseradish peroxidase for 1 hour at room temperature for 1 hour. The band was photographed by emitting light using chemical fluorescence (ECLPlus, GE Healthcare). The ratio of each group when the GAPDH expression level was 1 was calculated. An example of the results of the above test is shown in FIG.

  The expression enhancement of CSGANACT1 protein was recognized by addition of this extract of 0.1 mNU / mL (FIG. 1 and Table 6).

From the above, preferred embodiments of the present invention include the following, but are not limited thereto.

(1) An N-acetylgalactosamine transferase expression promoter containing an extract of inflamed tissue inoculated with vaccinia virus.
(2) The expression promoter according to (1), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.
(3) The expression promoter according to (1), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.
(4) The expression promoting action of N-acetylgalactosamine transferase 1 is stronger than the expression promoting action of N-acetylgalactosamine transferase 2 according to any one of (1) to (3) Expression promoter.
(5) The expression promoter according to any one of (1) to (4), wherein the inflamed tissue is an inflamed rabbit skin tissue.
(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 preparation.

(8) A method for judging or evaluating a vaccinia virus-inoculated inflammatory tissue extract or a preparation containing the same, using as an index the expression promoting action of N-acetylgalactosamine transferase in intervertebral disc cells.
(9) The determination or evaluation method according to (8), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.
(10) The determination or evaluation method according to (8), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.
(11) Compare the increase in expression of N-acetylgalactosamine transferase 1 and N-acetylgalactosamine transferase 2, and confirm that the increase in expression of N-acetylgalactosamine transferase 1 is greater (8) to The determination or evaluation method according to any one of (10).
(12) The determination or evaluation method according to any one of (8) to (11), wherein the inflamed tissue is rabbit inflamed skin tissue.

(13) A method for guaranteeing quality standards of an extract of inflamed tissue inoculated with vaccinia virus or a preparation containing the same by performing the determination or evaluation according to any of (8) to (12) above.
(14) The method for ensuring the quality standard according to (13) above, wherein the preparation is an injection or an oral preparation.

(15) Use of an extract of inflamed tissue inoculated with vaccinia virus for producing an N-acetylgalactosamine transferase expression promoter.
(16) The use according to (15), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.
(17) The use according to (15), wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.
(18) The N-acetylgalactosamine transferase expression promoter is characterized in that the N-acetylgalactosamine transferase 1 expression promoting action is stronger than the N-acetylgalactosamine transferase 2 expression promoting action (15 ) To (17).
(19) The use according to any one of (15) to (18), wherein the inflamed tissue is rabbit inflamed skin tissue.
(20) The use according to any one of (15) to (19), wherein the N-acetylgalactosamine transferase promoter is an injection.
(21) The use according to any one of (15) to (19), wherein the N-acetylgalactosamine transferase expression promoter is an oral agent.

  As described above, this extract has an action of promoting the expression of N-acetylgalactosamine transferase, particularly an action of accelerating the expression of N-acetylgalactosamine transferase 1 more strongly than N-acetylgalactosaminyltransferase 2. Therefore, the N-acetylgalactosamine transferase expression promoter containing this extract is useful as a therapeutic or preventive agent for diseases associated with intervertebral disc degeneration and OA.

Claims (14)

  N-acetylgalactosamine transferase expression promoter containing inflammatory tissue inoculated with vaccinia virus.   The expression promoter according to claim 1, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.   The expression promoter according to claim 1, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.   The expression promoting agent according to any one of claims 1 to 3, wherein the expression promoting action of N-acetylgalactosamine transferase 1 is stronger than the promoting action of N-acetylgalactosamine transferase 2 expression. .   The expression promoter according to any one of claims 1 to 4, wherein the inflamed tissue is an inflamed rabbit skin tissue.   The expression promoter according to any one of claims 1 to 5, which is an injection.   The expression promoter according to any one of claims 1 to 5, which is an oral preparation.   A method for determining or evaluating an extract of inflamed tissue inoculated with vaccinia virus or a preparation containing the same, using as an index the action of promoting the expression of N-acetylgalactosamine transferase in intervertebral disc cells.   The determination or evaluation method according to claim 8, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 1.   The determination or evaluation method according to claim 8, wherein the N-acetylgalactosamine transferase is N-acetylgalactosamine transferase 2.   The expression increase rate of N-acetylgalactosamine transferase 1 is compared with that of N-acetylgalactosamine transferase 2, and it is confirmed that the increase rate of expression of N-acetylgalactosamine transferase 1 is larger. The determination or evaluation method according to claim 1.   The determination or evaluation method according to any one of claims 8 to 11, wherein the inflamed tissue is an inflamed rabbit skin tissue.   A method for guaranteeing quality standards of an extract of inflamed tissue inoculated with vaccinia virus or a preparation containing the same by performing the determination or evaluation according to any one of claims 8 to 12.   The method for ensuring quality standards according to claim 13, wherein the preparation is an injection or an oral preparation.

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