AGENT FOR PROMOTING EXPRESSION OF N-ACETYLGALACTOSAMINYLTRANSFERASE CONTAINING EXTRACT FROM INFLAMED TISSUES INOCULATED WITH VACCINIA VIRUS

Abstract

An object of the present invention is to provide an agent for promoting expression of N-acetylgalactosaminyltransferase that contains an extract from inflamed tissues inoculated with vaccinia virus. The present invention demonstrated that the extract from inflamed tissues inoculated with vaccinia virus promotes the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells. Thus, the extract from inflamed tissues inoculated with vaccinia virus or a preparation containing the extract is useful as an agent for promoting the expression of N-acetylgalactosaminyltransferase in intervertebral disc cells.

Claims

1. A method for determining or evaluating an extract from inflamed tissues inoculated with vaccinia virus or a preparation containing the extract, wherein the action of promoting expression of an N-acetylgalactosaminyltransferase in intervertebral disc cells is used as an index.

2. The method according to claim 1, wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1.

3. The method according to claim 1, wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2.

4. The method according to claim 1, comprising comparing expression increase rates between the N-acetylgalactosaminyltransferase 1 and the N-acetylgalactosaminyltransferase 2, and confirming that the N-acetylgalactosaminyltransferase 1 has a higher expression increase rate.

5. The method according to claim 1, wherein the inflamed tissues are inflamed skin tissues of rabbits.

6. A method for verifying that the extract from inflamed tissues inoculated with vaccinia virus or the preparation satisfies the quality standard by performing the determination or evaluation of claim 1.

7. The method according to claim 6, wherein the preparation is an injectable preparation or an oral preparation.

Description

EXAMPLES

Example 1: Manufacture of the Present Extract

[0025] Skins of healthy adult rabbits were inoculated with vaccinia virus intradermally and the inflamed skins were cut and collected. The collected skins were washed and disinfected by a phenol solution, an excessive phenol solution was removed and the residue was crushed. A phenol solution was added thereto and mixed therewith and the mixture was allowed to stand for 3 to 7 days, and further heated at 35 to 40° C. together with stirring for 3 to 4 days. After that, an extracted solution obtained by a solid-liquid separation was adjusted to pH 4.5 to 5.2 with hydrochloric acid, heated at 90 to 100° C. for 30 minutes and filtered to remove protein. The filtrate was adjusted to pH 9.0 to 9.5 with sodium hydroxide, heated at 90 to 100° C. for 15 minutes and subjected to a solid-liquid separation.

[0026] The resulting deproteinized solution was adjusted to pH 4.0 to 4.3 with hydrochloric acid, activated carbon in an amount of 2% to the mass of the deproteinized solution was added thereto and the mixture was stirred for 2 hours and subjected to the solid-liquid separation. Water was added to the collected activated carbon followed by adjusting to pH 9.5 to 10 with sodium hydroxide and the mixture was stirred at 60° C. for 90 to 100 minutes and centrifuged to give a supernatant. Water was added again to the activated carbon precipitated upon the centrifugation followed by adjusting to pH 10.5 to 11 with sodium hydroxide and the mixture was stirred at 60° C. for 90 to 100 minutes and centrifuged to give a supernatant. Both supernatants were combined and neutralized with hydrochloric acid to give the present extract.

Example 2: Test Method and Test Results

[0027] The following describes the method of a pharmacological test conducted for demonstrating the GalNAcT expression promotion action in intervertebral disc cells due to the present extract obtained in Example 1, as well as the test results.

Cell and Reagent

[0028] Test Examples 1 to 3 used human nucleus pulposus cells prepared in accordance with the following procedure approved by the Experimentation Ethics Committee of Tokai University School of Medicine. Nucleus pulposus tissue was harvested from five patients with spinal disc herniation (three males and two females, aged 29 to 38) during surgery with their consent. The nucleus pulposus tissue was cut into fragments and 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 with an α-MEM medium (Wako Chemical) twice and seeded at a density of about 5×10.sup.3/cm.sup.2. The cells were cultured in an α-MEM medium containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin (Gibco), and 100 mg/ml streptomycin (Gibco) under 2% O.sub.2 (low oxygen) and 5% CO.sub.2 at 37° C. The medium was replaced twice a week, and the cells were treated with trypsin (Gibco) before reaching confluence to perform subculture. The cells obtained from the third subculture were used in the Examples.

[0029] The nucleus pulposus cells were seeded in a 25-cm.sup.2 flask at a density of 5000/cm.sup.2 and cultured in an α-MEM medium containing 10% FBS overnight before the present extract was added. Thereafter, the cells were treated with an α-MEM medium containing the present extract, 10% FBS, and 170 μM of ascorbic acid. While the culture was maintained for 2 weeks, the medium was replaced every other day. Nucleus pulposus cells that reached confluence were used in the following tests.

Statistical Analysis

[0030] Statistical analysis was performed with a repeated measures ANOVA. When the p value was less than 0.05, Bonferroni correction was performed.

Test Example 1: Effect on GAG Expression

GAG and DNA Analysis

[0031] The cultured cells were washed with Dulbecco's phosphate-buffered saline (DPBS, DS-Pharma) and treated with a buffer solution 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) at 65° C. overnight. The sulfated GAG content was calculated by measuring the absorbance at 656 nm using 1,9-dimethylmethylene blue (Biocolor), with chondroitin-6-sulfate (Biocolor) as the standard, with a SPECTRA MAX i3 spectrophotometer (Molecular Devices). The amount of DNA was calculated by a PicoGreen assay (ThermoFisher Scientific, Waltham, Mass.) with excitation at 480 nm and luminescence at 520 nm, using the same spectrophotometer. The ratio of GAG to the amount of DNA (average value±standard error of the mean) was calculated. Table 1 illustrates some of the results of this test.

TABLE-US-00001 TABLE 1 The Ratio of GAG to DNA Control 4.33 ± 0.38 Ascorbic Acid 5.88 ± 0.73 Ascorbic Acid  7.12 ± 1.20** Present Extract 0.1 mNU/mL Ascorbic Acid 6.05 ± 0.76 Present Extract 1.0 mNU/mL **p < 0.01 vs Control

[0032] The value of GAG/DNA increased 1.7 times with the group administered the present extract in an amount of 0.1 mNU/mL, and 1.4 times with the group administered the present extract in an amount of 1.0 mNU/mL, compared to that of the control group, exhibiting significant enhancement of GAG production in the group administered the present extract in an amount of 0.1 mNU/mL (Table 1).

Test Example 2: Effect on Expression of CSGALNACT1, ANG1, or IGF Gene (Quantitative Real-Time PCR)

[0033] After 1 week from the addition of the present extract in an amount of 1.0 mNU/mL, the cells were collected and homogenized in a lysis buffer. Total RNA (tRNA) was then prepared using an SV Total RNA isolation system (Promega). 2 μg of tRNA of each sample was reverse-transcribed into cDNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems). The amount of mRNA of GALNACT1 was calculated by the comparative CT method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH: trade name, pre-developed TaqMan Assay Reagents (Applied Biosystems)) as the internal standard. The following primer and probe (Applied Biosystems) were used: IGF1 (TaqMan Assay ID: Hs03986524_m1), ANGPT1 (TaqMan Assay ID: Hs00181613_m1), and CSGALNACT1 (TaqMan Assay ID: Hs00218054_m1). With the expression level of CSGALNACT1, ANGPT1, or IGF1 in the control taken as 1, the ratio of each group (average value±standard error of the mean) was calculated. Tables 2 to 4 illustrate some of the results of this test.

TABLE-US-00002 TABLE 2 Ratio to Expression Level of CSGALNACT1 mRNA in Control Taken as 1 Control 1.00 Ascorbic Acid 2.25 ± 0.55  Ascorbic Acid 3.95 ± 1.25** Present Extract 0.1 mNU/mL Ascorbic Acid 2.53 ± 0.43*  Present Extract 1.0 mNU/mL *p < 0.05 vs Control, **p < 0.01 vs Control

TABLE-US-00003 TABLE 3 Ratio to Expression Level of ANGPT1 mRNA in Control Taken as 1 Control 1.00 Ascorbic Acid 3.80 ± 0.91** Ascorbic Acid 6.91 ± 1.80** Present Extract 0.1 mNU/mL Ascorbic Acid 5.44 ± 1.09** Present Extract 1.0 mNU/mL **p < 0.01 vs Control

TABLE-US-00004 TABLE 4 Ratio to Expression Level of IGF1 mRNA in Control Taken as 1 Control 1.00 Ascorbic Acid 8.92 ± 3.30** Ascorbic Acid 12.7 ± 3.22** Present Extract 0.1 mNU/mL Ascorbic Acid 9.83 ± 2.17** Present Extract 1.0 mNU/mL **p < 0.01 vs Control

[0034] The expression level of CSGALNACT1 was confirmed to have been significantly enhanced by adding the present extract in an amount of 0.1 mNU/mL or 1.0 mNU/mL (Table 2). The expression level of ANGPT1 mRNA and the expression level of IGF1 mRNA were also confirmed to have been significantly enhanced by adding the present extract in an amount of 0.1 mNU/mL or 1.0 mNU/mL (Tables 3 and 4).

Test Example 3: Effect on Expression of CSGALNACT1 mRNA and CSGALNACT2 mRNA (Microarray Analysis)

[0035] Gene expressions in nucleus pulposus cells derived from a patient—nucleus pulposus cells treated with the present extract in an amount of 1.0 mNU/mL and untreated nucleus pulposus cells—were compared using a microarray. The present extract and 170 μM of ascorbic acid were added to the cells. tRNA was prepared in the same manner as in Test Example 2, and Cy3-labeled cRNA was prepared using an Low Input Quick Amp Labeling Kit (Agilent Technology). The obtained Cy3-labeled cRNA was subjected to hybridization using a SurePrint G3 Human GE 8×60K v2

[0036] Microarray (Agilent Technology) and a Gene Expression Hybridization Kit (Agilent Technology). Thereafter, analysis was performed with an Agilent DNA microarray scanner (Agilent Technology, G2600D SG13164306) in accordance with the AgilentG3_HiSen_GX_1Color (Agilent Technology) protocol.

[0037] The fluorescence intensity of each probe was converted to an expression level using Agilent Feature Extraction 11.5.1.1 digitization conversion software (Agilent Technology). Genes expressed at high levels were detected using Gene Spring ver.13 gene expression analysis software (Agilent Technology). Genes involved in GAG synthesis were selected using the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2017 Tool, and Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY Database. With the expression level of a sample that was cultured for the same time period without the present extract (control) taken as 1, the ratio of the expression level after addition of the present extract was calculated. The average of signals for the same gene emitted from multiple probes in a microarray was calculated and used as one piece of data. Table 5 illustrates some of the results of this test.

TABLE-US-00005 TABLE 5 Ratio to Expression Gene Name Level of Control Taken (Enzyme Name) as 1 Control 1.00 CSGALNACT1 1.54 (chondroitin sulfate N- acetylgalactosaminyltransferase 1) HS3ST3A1 1.45 (heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A) EXTL1 1.41 (heparan sulfate GlcA/GlcNAc transferase like 1) CHSY3 1.37 (N-Acetylgalactosaminyltransferase Glucuronyltransferase) GLCE 1.25 (glucuronic acid epimerase) HS3ST5 1.19 (heparan sulfate 3-OST-5) HS3ST3B1 1.18 (heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3B) HS6ST1 1.16 (heparan sulfate 6-O- sulfotransferase) FUT8 1.14 (alpha 1,6-fucosyltransferase 8) HS3ST1 1.13 (heparan sulfate D-glucosaminyl 3-O-sulfotransferase) CHST6 1.13 (corneal N-acetylglucosamine 6- sulfotransferase) CSGALNACT2 1.12 (chondroitin sulfate N- acetylgalactosaminyltransferase 2) CHST13 1.12 (chondroitin D-N- acetylgalactosamine-4-O- sulfotransferase 3) CHST12 1.09 (chondroitin D-N- acetylgalactosamine-4-O- sulfotransferase 2) XYLT2 1.08 (xylosyltransferase 2)
(continued from Table 5)

[0038] In most genes involved in GAG synthesis, enhancement of the expression due to the addition of the present extract in an amount of 1.0 mNU/mL was observed (Table 5). The expression of CSGALNACT was enhanced by 1.54 times in CSGALNACT1 and 1.12 times in CSGALNACT2 (Table 5).

[0039] The preferable embodiments of the present invention thus include, but are not limited to, the following subject matter.

(1) An agent for promoting expression of an N-acetylgalactosaminyltransferase containing an extract from inflamed tissues inoculated with vaccinia virus.
(2) The agent according to (1), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1.
(3) The agent according to (1), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2.
(4) The agent according to any of (1) to (3), wherein the agent has a higher expression-promoting action on the N-acetylgalactosaminyltransferase 1 than on the N-acetylgalactosaminyltransferase 2.
(5) The agent according to any of (1) to (4), wherein the inflamed tissues are inflamed skin tissues of rabbits.
(6) The agent according to any of (1) to (5), wherein the agent is an injectable preparation.
(7) The agent according to any of (1) to (5), wherein the agent is an oral preparation.
(8) A method for determining or evaluating an extract from inflamed tissues inoculated with vaccinia virus or a preparation containing the extract, wherein the action of promoting expression of an N-acetylgalactosaminyltransferase in intervertebral disc cells is used as an index.
(9) The method according to (8), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 1.
(10) The method according to (8), wherein the N-acetylgalactosaminyltransferase is N-acetylgalactosaminyltransferase 2.
(11) The method according to any of (8) to (10), comprising

[0040] comparing expression increase rates between the N-acetylgalactosaminyltransferase 1 and the N-acetylgalactosaminyltransferase 2, and

[0041] confirming that the N-acetylgalactosaminyltransferase 1 has a higher expression increase rate.

(12) The method according to any of (8) to (11), wherein the inflamed tissues are inflamed skin tissues of rabbits.
(13) A method for verifying that the extract from inflamed tissues inoculated with vaccinia virus or the preparation satisfies the quality standard by performing the determination or evaluation of any of (8) to (12).
(14) The method according to (13), wherein the preparation is an injectable preparation or an oral preparation.
(15) Use of an extract from inflamed tissues inoculated with vaccinia virus in the production of an agent for promoting expression of an N-acetylgalactosaminyltransferase.
(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 of (15) to (17), wherein the agent for promoting expression of an N-acetylgalactosaminyltransferase has a higher expression-promoting action on the N-acetylgalactosaminyltransferase 1 than on the N-acetylgalactosaminyltransferase 2.
(19) The use according to any of (15) to (18), wherein the inflamed tissues are inflamed skin tissues of rabbits.
(20) The use according to any of (15) to (19), wherein the agent for promoting expression of an N-acetylgalactosaminyltransferase is an injectable preparation.
(21) The use according to any of (15) to (19), wherein the agent for promoting expression of an N-acetylgalactosaminyltransferase is an oral preparation.

INDUSTRIAL APPLICABILITY

[0042] As described above, the present extract has action for promoting the expression of N-acetylgalactosaminyltransferases, in particular action for promoting the expression of N-acetylgalactosaminyltransferase 1 more than the expression of N-acetylgalactosaminyltransferase 2. This indicates that an agent for promoting the expression of an N-acetylgalactosaminyltransferase containing the present extract is useful as a therapeutic or preventive preparation for diseases associated with intervertebral disc degeneration or osteoarthritis.