U.S. patent application number 13/821617 was filed with the patent office on 2014-09-11 for method for promoting the synthesis of collagen and proteoglycan in chondrocytes.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. The applicant listed for this patent is Koichi Masuda, Mitsuru Naiki. Invention is credited to Koichi Masuda, Mitsuru Naiki.
Application Number | 20140255507 13/821617 |
Document ID | / |
Family ID | 45938923 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255507 |
Kind Code |
A9 |
Masuda; Koichi ; et
al. |
September 11, 2014 |
METHOD FOR PROMOTING THE SYNTHESIS OF COLLAGEN AND PROTEOGLYCAN IN
CHONDROCYTES
Abstract
The synthesis of collagen and proteoglycan in chondrocytes, such
as intervertebral disc cells, articular chondrocytes and meniscal
cells is promoted by administration of an extract from inflamed
tissue inoculated with vaccinia virus.
Inventors: |
Masuda; Koichi; (San Diego,
CA) ; Naiki; Mitsuru; (Katoh-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Masuda; Koichi
Naiki; Mitsuru |
San Diego
Katoh-shi |
CA |
US
JP |
|
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
NIPPON ZOKI PHARMACEUTICAL CO., LTD.
Osaka
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130164383 A1 |
June 27, 2013 |
|
|
Family ID: |
45938923 |
Appl. No.: |
13/821617 |
Filed: |
October 11, 2011 |
PCT Filed: |
October 11, 2011 |
PCT NO: |
PCT/US11/55757 PCKC 00 |
371 Date: |
March 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61393190 |
Oct 14, 2010 |
|
|
|
Current U.S.
Class: |
424/574 ;
435/29 |
Current CPC
Class: |
A61P 19/00 20180101;
A61P 19/08 20180101; A61P 29/00 20180101; C12Q 1/025 20130101; A61P
9/00 20180101; A61K 35/32 20130101; A61K 35/36 20130101; G01N
33/5082 20130101; A61K 35/76 20130101; A61P 25/04 20180101; G01N
2333/07 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/574 ;
435/29 |
International
Class: |
A61K 35/36 20060101
A61K035/36 |
Claims
1. A method for promoting the synthesis of proteoglycan and/or
collagen in chondrocytes comprising administering to a patient in
need of such treatment an extract isolated from inflamed tissue
inoculated with vaccinia virus.
2. A method as claimed in claim 1 wherein said chondrocytes
comprise intervertebral disc cells.
3. A method as claimed in claim 2 wherein said intervertebral disc
cells comprise bovine nucleus pulposus cells and/or anulus fibrosus
cells.
4. A method as claimed in claim 2 wherein proteoglycan synthesis is
promoted.
5. A method as claimed in claim 2 wherein collagen synthesis is
promoted.
6. A method as claimed in claim 2 wherein the inflamed tissue is a
rabbit skin tissue.
7. A method as claimed in claim 4 wherein said intervertebral disc
cells comprise bovine nucleus pulposus cells.
8. A method as claimed in claim 4 wherein said intervertebral disc
cells comprise anulus fibrosus cells.
9. A method as claimed in claim 5 wherein said intervertebral disc
cells comprise bovine nucleus pulposus cells.
10. A method as claimed in claim 5 wherein said intervertebral disc
cells comprise anulus fibrosus cells.
11. A method for determining or evaluating an extract isolated from
inflamed tissue inoculated with vaccinia virus comprising measuring
a promoting activity on the synthesis of proteoglycan and/or
collagen in cultured chondrocytes by said extract as an index.
12. A method as claimed in claim 11 wherein said chondrocytes
comprise intervertebral disc cells.
13. A method as claimed in claim 12 wherein said intervertebral
disc cells comprise bovine nucleus pulposus cells and/or anulus
fibrosus cells.
14. A method as claimed in claim 12 wherein proteoglycan synthesis
is promoted.
15. A method as claimed in claim 12 wherein collagen synthesis is
promoted.
16. A method as claimed in claim 12 wherein the inflamed tissue is
a rabbit skin tissue.
17. A method for regenerating a chondrocyte extracellular matrix,
comprising administering to a patient in need of such treatment an
extract isolated from inflamed tissue inoculated with vaccinia
virus.
18. A method as claimed in claim 17 wherein the chondrocyte
extracellular matrix is an intervertebral disc cell extracellular
matrix.
19. A method for determining or evaluating an extract isolated from
inflamed tissue inoculated with vaccinia virus comprising measuring
a regenerating activity of a chondrocyte extracellular matrix by
said extract as an index.
20. A method as claimed in claim 19 wherein said chondrocyte
extracellular matrix is an intervertebral disc cell extracellular
matrix.
21. A method for treating degenerative intervertebral discs,
comprising administering to a patient in need of such treatment an
extract isolated from inflamed tissue inoculated with vaccinia
virus.
22. A method for treating pain caused by intervertebral disc
degeneration, comprising regenerating an intervertebral disc cell
extracellular matrix by administering to a patient in need of such
treatment an extract isolated from inflamed tissue inoculated with
vaccinia virus.
23. A method for treating a pain caused by intervertebral disc
degeneration comprising promoting the synthesis of proteoglycan in
intervertebral disc cells by administering to a patient in need of
such treatment an extract isolated from inflamed tissue inoculated
with vaccinia virus.
24. A method for treating pain caused by intervertebral disc
degeneration, comprising promoting the synthesis of collagen in
intervertebral disc cells by administering to a patient in need of
such treatment an extract isolated from inflamed tissue inoculated
with vaccinia virus.
25. An agent for treating or preventing degenerative intervertebral
discs comprising an extract isolated from inflamed tissue
inoculated with vaccinia virus.
26. An agent for promoting the synthesis of proteoglycan and/or
collagen in intervertebral disc cells comprising an extract
isolated from inflamed tissue inoculated with vaccinia virus.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel medical use of an
extract from inflamed tissue inoculated with vaccinia virus, and in
particular, relates to a method for promoting the synthesis of
collagen and proteoglycan in chondrocytes, such as intervertebral
disc cells, articular chondrocytes and meniscal cells.
BACKGROUND OF THE INVENTION
[0002] A medical agent, which has been sold under the brand name of
"Neurotropin", contains a nonprotein extract from the inflamed skin
of rabbits following inoculation with vaccinia virus as an active
ingredient. Neurotropin (NTP) has been widely used to treat
neuropathic pain, including post-herpetic neuralgia and low back
pain in Japan. In animal models, NTP has been shown that the
anti-nociceptive effect was derived by the activation of the
descending monoaminergic pain inhibitory system. NTP has also been
shown to inhibit the pain pathway by inhibiting activation of the
kallikrein-kinin cascade and, consequently, the formation of
bradykinin in vitro and in vivo.
[0003] The suppressive effect of NTP on tumor necrosis
factor-.alpha.(TNF-.alpha.) and cyclooxygenase-2 (COX-2) mRNA
expression by human intervertebral disc cells was recently
reported. However, it is not clear if NTP has an effect on
intervertebral disc cell extracellular matrix (ECM) synthesis.
SUMMARY OF THE INVENTION
[0004] An extract from inflamed tissue inoculated with vaccinia
virus is employed to promote the synthesis of collagen and
proteoglycan in chondrocytes, such as intervertebral disc cells,
such as bovine nucleus pulposus cells or annulus fibrosus cells,
articular chondrocytes and meniscal cells. In an aspect of the
invention, there is provided a method for promoting the synthesis
of proteoglycan and/or collagen in chondrocytes in a patient,
comprising administering to a patient in need of such treatment a
pharmaceutically effective amount of an extract isolated from
inflamed tissue inoculated with vaccinia virus. In another aspect
of the present invention there is provided a method for
regenerating a chondrocyte extracellular matrix, comprising
administering to a patient in need of such treatment an extract
isolated from inflamed tissue inoculated with vaccinia virus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The present invention is further illustrated by the
accompanying drawings wherein:
[0006] FIG. 1 is an experimental result of an activity on the
synthesis of proteoglycan in intervertebral disc cells which are
anulus fibrosus cells (AF) of an extract from inflamed tissue
inoculated with vaccinia virus.
[0007] FIG. 2 is an experimental result of an activity on the
synthesis of proteoglycan in intervertebral disc cells which are
bovine nucleus pulposus cells (NP) of an extract from inflamed
tissue inoculated with vaccinia virus.
[0008] FIG. 3 is an experimental result of an activity on the
synthesis of collagen in intervertebral disc cells which are anulus
fibrosus cells (AF) of an extract from inflamed tissue inoculated
with vaccinia virus.
[0009] FIG. 4 is an experimental result of an activity on the
synthesis of collagen in intervertebral disc cells which are bovine
nucleus pulposus cells (NP) of an extract from inflamed tissue
inoculated with vaccinia virus.
DETAILED DESCRIPTION OF THE INVENTION
[0010] This invention is based on the results to assess the effects
of NTP on proteoglycan (PG) and collagen synthesis using bovine
nucleus pulposus (NP) and anulus fibrosus (AF) cells cultured in
alginate beads under normoxic and hypoxic conditions.
[0011] As for the extract from inflamed tissue inoculated with
vaccinia virus of the present invention, there are various reports
on physiological active substances produced in the inflamed tissue
inoculated with vaccinia virus, the method for extracting the
substances from the diseased tissue, the pharmacological activities
and the like as previously reported (see paragraph [0008] of WO
2009/028605, and EP2191836, and Japanese Patent Application
Publication Nos. JP-A-53-101515, JP-A-55-87724, JP-A-1-265028,
JP-A-1-319422, JP-A-2-28119, JP-A-7-97336, JP-A-8-291077,
JP-A-10-194978, JP-A-11-80005, JP-A-11-139977, JP-A-2000-336034,
JP-A-2000-16942, and JP-A-2004-300146, and International
Publication No. WO 2004/039383.)
[0012] Furthermore, a preparation of an extract from inflamed skins
of rabbits inoculated with vaccinia virus is commercially available
as a pharmaceutical product, and may be employed in the present
invention. The preparation, as described in pages 2978 to 2980 of
"Drugs in Japan, Ethical Drugs" (2010, edited and published by
Japan Pharmaceutical Information Center), is a medicinal agent
containing non-proteinous active substances extracted and separated
from the inflamed skin tissue of rabbits inoculated with vaccinia
virus. The preparation is known to be effective against low back
pain, cervicobrachial syndrome, symptomatic neuralgia,
periarthritis scapulohumeralis, osteoarthritis, itchiness
accompanied with skin diseases (eczema, dermatitis, urticaria),
allergic rhinitis, sequelae of subacute myelo-optico-neuropathy
such as coldness, paresthesia and pain, postherpetic neuralgia and
the like. The preparation is approved as an ethical drug in the
form of hypodermic, intramuscular and intravenous injection
products and of tablets and is commercially available.
[0013] The extract from inflamed tissue inoculated with vaccinia
virus used in the present invention is a non-proteinous
biofunction-regulating substance extracted from the inflamed tissue
inoculated with vaccinia virus as described above, and the
preparation of the extracted solution from inflamed skins of
rabbits inoculated with vaccinia virus listed in the "Drugs in
Japan, Ethical Drugs" is approved as a pharmaceutical product and
is commercially available. In addition, various extracts from
inflamed tissue inoculated with vaccinia virus described in Patent
Documents described above may be used as the substance of the
present invention, and their producing methods, suitable doses and
the like are also given in the documents.
[0014] The extract from inflamed tissue inoculated with vaccinia
virus of the present invention can be obtained in the following
manner: inflamed tissue inoculated with vaccinia virus is crushed;
an extraction solvent is added to remove the tissue fragments; then
deproteinization is carried out; the deproteinized solution is
adsorbed onto an adsorbent; and then the active ingredient is
eluted.
[0015] The extract from inflamed tissue inoculated with vaccinia
virus is produced, for example, according to the following
process.
[0016] (a) Inflamed skin tissues of rabbits, mice or the like by
the inoculation with vaccinia virus are collected, and the inflamed
tissues are crushed. To the crushed tissue an extraction solvent
such as water, phenolated water, physiological saline or
phenol-added glycerin water is added. Then, the mixture is filtered
or centrifuged to obtain an extraction liquid (filtrate or
supernatant).
[0017] (b) The pH of the extraction liquid is adjusted to an acidic
condition and the liquid is heated for deproteinization. Then, the
deproteinized solution is adjusted to an alkaline condition,
heated, and then filtered or centrifuged.
[0018] (c) The obtained filtrate or supernatant is made acidic and
adsorbed onto an adsorbent such as activated carbon or kaolin.
[0019] (d) To the adsorbent, an extraction solvent such as water is
added, the pH is adjusted to an alkaline condition, and the
adsorbed component is eluted to obtain the extract from inflamed
tissues inoculated with vaccinia virus. Subsequently, as desired,
the eluate may be evaporated to dryness under reduced pressure or
freeze-dried to give a dried material.
[0020] As for animals in order to obtain the inflamed tissues by
the inoculation of vaccinia virus, various animals that are
infected with vaccinia virus such as rabbits, cows, horses, sheep,
goats, monkeys, rats or mice can be used, and preferred inflamed
tissues are inflamed skin tissues of rabbits.
[0021] The inflamed tissues are collected and crushed, and 1 to 5
volumes of extraction solvent is added to make an emulsified
suspension. As for the extraction solvent, distilled water,
physiological saline, weakly acidic to weakly basic buffer and the
like can be used, and stabilizers such as glycerin,
antibacterial/antiseptic agents such as phenol, and salts such as
sodium chloride, potassium chloride or magnesium chloride may be
suitably added. At this time, the extraction may be facilitated by
breaking the cellular tissues with treatment such as freezing and
thawing, ultrasonic waves, cell membrane dissolving enzymes or
surfactants.
[0022] The obtained emulsified extraction liquid is subjected to
filtration, centrifugation or the like to remove tissue fragments,
and then deproteinized. The deproteinization operation may be
carried out by a generally known method, for example, heat
treatment, treatment with a protein denaturant such as an acid,
base, urea and guanidine, treatment with an organic solvent such as
acetone, isoelectric precipitation, and salting out can be applied.
Then, by a general method for removing insolubles such as
filtration using filter paper (for example, cellulose or
nitrocellulose), glass filters, Celite, Seitz filters or the like,
ultrafiltration and centrifugation, the precipitated insoluble
protein is removed.
[0023] The extraction liquid containing active ingredients obtained
in this manner is acidified, preferably adjusted to pH 3.5 to 5.5
with an acid such as hydrochloric acid, sulfuric acid or
hydrobromic acid, and then adsorbed onto an adsorbent. Examples of
the usable adsorbent include activated carbon and kaolin. The
adsorbent may be added into the extraction liquid with stirring, or
the extraction liquid may be passed through a column filled with
the adsorbent to adsorb the active ingredients onto the adsorbent.
When the adsorbent is added into the extraction liquid, the
solution is removed by filtration, centrifugation, or the like to
obtain the adsorbent in which the active ingredients are
adsorbed.
[0024] In order to elute (desorb) the active ingredients from the
adsorbent, an elution solvent is added to the adsorbent to elute at
room temperature or with suitable heating or with stirring, and the
adsorbent is removed by a general method such as filtration,
centrifugation, or the like. As for the elution solvent to be used,
a basic solvent such as water, methanol, ethanol or isopropanol
that are adjusted to have a basic pH or a suitable mixture thereof
may be used, and preferably water adjusted to pH 9 to 12 may be
used.
[0025] The extract (eluate) obtained in this manner may be properly
prepared in a suitable form as a raw material for a formulation or
a pharmaceutical formulation. For example, the solution may be
adjusted to have a nearly neutral pH to be a raw material for a
formulation, and may be adjusted to have a desired concentration by
concentration or dilution. In addition, for a formulation for
injection, sodium chloride may be added to prepare a solution
isotonic to physiological saline. Furthermore, the solution may be
concentrated to dryness or freeze-dried to prepare a solid form
available for the raw material of tablets or the like.
[0026] Examples of an administration method to a patient in need of
treatment include oral and other administrations such as
subcutaneous, intramuscular and intravenous administrations, in
pharmaceutically effective amounts. The dose can be suitably
determined depending on the type of extract from inflamed tissues
inoculated with vaccinia virus. The dose that is approved in the
commercially available preparation according to the "Drugs in
Japan, Ethical Drugs" (page 2978) is principally 16 NU per day by
oral administration and 3.6 to 7.2 NU per day by injection as an
ethical drug. However, the dose may be appropriately increased or
decreased depending on the type of disease, degree of seriousness,
individual difference in the patients, method of administration,
period of administration and the like (NU: Neurotropin unit.
Neurotropin unit is defined by ED.sub.50 value of analgesic effect
measured by a modified Randall-Selitto method using SART-stressed
mice that are chronic stressed animals showing a lowered pain
threshold than normal animals. One NU indicates the activity of 1
mg of analgesic ingredients in Neurotropin preparations when the
ED.sub.50 value is 100 mg/kg of the preparation).
[0027] Hereinafter presented are examples of methods for producing
an extract from inflamed tissues inoculated with vaccinia virus as
well as results of a pharmacological test concerning novel
pharmacological activity of the extract, that is, the promoting
activity on the synthesis of collagen and proteoglycan in
intervertebral disc cells. The present invention is not intended to
be limited to the descriptions in the following Examples, where all
parts, percentages, and ratios are by weight, all temperatures are
in .degree. C., and all pressures are atmospheric unless indicated
to the contrary:
EXAMPLE 1
[0028] Skins of healthy adult rabbits were inoculated with vaccinia
virus. The inflamed skins were removed and crushed, and to the
crushed skins, phenolated water was added. Then, the mixture was
filtered under pressure, and the obtained filtrate was adjusted to
pH 5 with hydrochloric acid, and then heated at 90 to 100.degree.
C. for 30 minutes. After deproteinization by filtration, the
filtrate was adjusted to pH 9 with sodium hydroxide, further heated
at 90 to 100.degree. C. for 15 minutes, and then filtered. The
filtrate was adjusted to about pH 4.5 with hydrochloric acid, and
2% activated carbon was added. The mixture was stirred for 2 hours
and then centrifuged. To the collected activated carbon, water was
added. The mixture was adjusted to pH 10 with sodium hydroxide,
stirred at 60.degree. C. for 1.5 hours, and then centrifuged and
filtered to obtain a supernatant. To the collected activated
carbon, water was added again. The mixture was adjusted to pH 11
with sodium hydroxide, stirred at 60.degree. C. for 1.5 hours, and
then centrifuged to obtain a supernatant. The two supernatants were
combined and neutralized with hydrochloric acid to obtain an
extract from inflamed skins of rabbits inoculated with vaccinia
virus. In the following pharmacological tests, the extract was
adjusted to an appropriate concentration to be used.
EXAMPLE 2
[0029] Skins of healthy adult rabbits were inoculated with vaccinia
virus to be infected. Subsequently, the inflamed skins were
aseptically removed and chopped, and then phenol-added glycerin
water was added. The mixture was ground with a homogenizer to be
emulsified. Subsequently, the emulsion was filtered. The obtained
filtrate was adjusted to weak acidity (pH 4.5 to 5.5) with
hydrochloric acid, then heated at 100.degree. C. and filtered. The
filtrate was adjusted to weak alkalinity (pH 8.5 to 10.0) with
sodium hydroxide, further heated at 100.degree. C. and then
filtered. The filtrate was adjusted to about pH 4.5 with
hydrochloric acid, and about 1.5% activated carbon was added. The
mixture was stirred for 1 to 5 hours and then filtered. To the
activated carbon collected by the filtration, water was added. The
mixture was adjusted to pH 9.4 to 10 with sodium hydroxide, stirred
for 3 to 5 hours, and then filtered. The filtrate was neutralized
with hydrochloric acid.
EXAMPLE 3
[0030] Next, an example of the pharmacological test results
concerning promoting activity on the synthesis of collagen and
proteoglycan in intervertebral disc cells in which the extract from
inflamed tissue inoculated with vaccinia virus (NTP) of the present
invention obtained in Example 1 was used as a test substance, is
shown. The effect of NTP on the metabolism of proteoglycan (PG) and
collagen synthesis and cell proliferation in intervertebral disk
cells is given below and in FIGS. 1-4.
[0031] 1. Materials and Methods
[0032] Bovine nucleus pulposus (NP) and anulus fibrosus (AF) cells,
isolated from 14-18 month-old bovine coccygeal intervertebral
discs, were encapsulated in alginate (4E+6 cells/mL) and cultured
for 11 days in DMEM/F12 with 10% fetal bovine serum (FBS) under
normoxic (21% O2) or hypoxic (5% O2) conditions. These cultures
were treated with three different concentrations (0.001 NU /ml,
0.01 NU/mL, and 0.1 NU/mL) of NTP for three days. To assess the
synthesis of PGs and collagen, the cultures were labeled with
35S-sulfate or .sup.3H-proline during the last 4 or 16 hours,
respectively. The beads and media were collected and digested by
papain after labeling, as previously described [Masuda, K. et al.,
J Orthop Res., 21, 922-930 (2003) and Akeda, K, Spine, 31, 959-966
(2006)], and the radioactive precursor incorporation was measured
in media and alginate beads separately. Cell proliferation was
assessed using the CellTiter 96 Aqueous One Solution Cell
Proliferation Assay (Promega, Madison, Wis.). All data from three
experiments from different batches of cells were normalized with
the average value of the control group in each experiment. The
effects of treatment were assessed using ANOVA with PSLD test as a
posthoc test.
[0033] 2. Results
[0034] 1) Cell Proliferation
[0035] The oxygen concentration and treatment with NTP did not
affect NP and AF cell proliferation at both the 7 and 14 days time
points.
[0036] 2) PG Synthesis
[0037] In NP cells, PG synthesis was significantly increased with
NTP treatment under culture at 5% O.sub.2 (at 0.01 NU/ml, +65%,
p<0.05, at 0.1 NU/ml, +66%, p<0.05, FIG. 2). There were no
significant differences in PG synthesis in NP cells under normoxic
culture. As shown in FIG. 1, in AF cells, NTP did not change the
level of PG synthesis under both normoxic and hypoxic
conditions.
[0038] 3) Collagen Synthesis
[0039] Collagen synthesis by NP cells in under 5% O.sub.2 and NP
and AF cells under 21% O.sub.2 was increased by treatment with NTP
in a dose-dependent manner, as shown in FIGS. 3 and 4 (maximum
stimulation at 0.1 NU/ml: NP, 5% O.sub.2, +50%; NP, 21% O.sub.2,
+63%; AF, 21% O.sub.2 +47%).
[0040] As shown above, this study has shown for the first time that
NTP can stimulate the extracellular matrix synthesis of
intervertebral disc cells. Interestingly, the effect of NTP on PG
synthesis by NP cells was more apparent under 5% O.sub.2 than under
21% O.sub.2 culture conditions. Considering the hypoxic condition
of the NP in vivo, these results may suggest that NP cells can
respond to NTP under physiologically relevant conditions. Although
the positive effects of NTP on collagen synthesis by NP cells were
observed in both 5% O.sub.2 and 21% O.sub.2, stimulation in the AF
was only observed in the 21% O.sub.2 condition. Unlike the
increased synthesis of PGs by NP cells in response to NTP, AF cells
were more responsive to NTP under the 21% O.sub.2 condition.
Because oxygen tension is significantly higher in the AF than in
the NP, the greater response of AF cells to NTP at 21% O.sub.2 may
also reflect optimal culture conditions.
[0041] Chondrocytes are present in various cartilages such as
articular cartilage, intervertebral disc, meniscus, etc. The
chondrocytes produce an extracellular matrix around them with cell
divisions to make cartilage formation. Cartilages are classified
into groups of hyaline cartilage, elastic cartilage and fibro
cartilage according to the content ratio of amorphous matrix and
fibrous material. The main components of the extracellular matrix
are collagen and proteoglycan. Since NTP has a promoting activity
on the synthesis of collagen and proteoglycan in chondrocytes such
as intervertebral disc cells, it is indicated that NTP has a
regenerating activity on a chondrocyte extracellular matrix of
various cartilages.
INDUSTRIAL APPLICABILITY
[0042] The present invention relates to a novel medical use of an
extract from inflamed tissue inoculated with vaccinia virus, and in
particular, relates to a method for promoting the synthesis of
collagen and proteoglycan in chondrocytes, such as intervertebral
disc cells, articular chondrocytes and meniscal cells. Preferred
embodiments of the present invention are given as follows:
[0043] 1. A method for promoting the synthesis of proteoglycan
and/or collagen in chondrocytes in a patient, comprising
administering to a patient in need of such treatment an extract
isolated from inflamed tissue inoculated with vaccinia virus.
[0044] 2. An agent for promoting the synthesis of proteoglycan
and/or collagen in chondrocytes containing an extract isolated from
inflamed tissue inoculated with vaccinia virus.
[0045] 3. A use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for promoting the synthesis of
proteoglycan and/or collagen in chondrocytes.
[0046] 4. A method for determining or evaluating an extract
isolated from inflamed tissue inoculated with vaccinia virus
wherein a promoting activity on the synthesis of proteoglycan
and/or collagen in cultured chondrocytes is used as an index.
[0047] 5. An extract isolated from inflamed tissue inoculated with
vaccinia virus having a promoting activity on the synthesis of
proteoglycan and/or collagen in chondrocytes.
[0048] 6. A method for regenerating a chondrocyte extracellular
matrix, comprising administering to a patient in need of such
treatment an extract isolated from inflamed tissue inoculated with
vaccinia virus.
[0049] 7. An agent for regenerating a chondrocyte extracellular
matrix containing an extract isolated from inflamed tissue
inoculated with vaccinia virus.
[0050] 8. Use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for regenerating a chondrocyte
extracellular matrix.
[0051] 9. A method for determining or evaluating an extract
isolated from inflamed tissue inoculated with vaccinia virus
wherein a regenerating activity of a chondrocyte extracellular
matrix is used as an index.
[0052] 10. An extract isolated from inflamed tissue inoculated with
vaccinia virus having a regenerating activity of a chondrocyte
extracellular matrix.
[0053] 11. A method for promoting the synthesis of proteoglycan in
intervertebral disc cells (bovine nucleus pulposus cells),
comprising administering to a patient in need of such treatment an
extract isolated from inflamed tissue inoculated with vaccinia
virus.
[0054] 12. A method for promoting the synthesis of collagen in
intervertebral disc cells (bovine nucleus pulposus cells or anulus
fibrosus cells), comprising administering to a patient in need of
such treatment an extract isolated from inflamed tissue inoculated
with vaccinia virus.
[0055] 13. An agent for promoting the synthesis of proteoglycan in
intervertebral disc cells (bovine nucleus pulposus cells)
containing an extract isolated from inflamed tissue inoculated with
vaccinia virus.
[0056] 14. An agent for promoting the synthesis of collagen in
intervertebral disc cells (bovine nucleus pulposus cells or anulus
fibrosus cells) containing an extract isolated from inflamed tissue
inoculated with vaccinia virus.
[0057] 15. Use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for promoting the synthesis of
proteoglycan in intervertebral disc cells (bovine nucleus pulposus
cells).
[0058] 16. Use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for promoting the synthesis of
collagen in intervertebral disc cells (bovine nucleus pulposus
cells or anulus fibrosus cells).
[0059] 17. A method for determining or evaluating an extract
isolated from inflamed tissue inoculated with vaccinia virus
wherein a promoting activity on the synthesis of proteoglycan in
cultured intervertebral disc cells (bovine nucleus pulposus cells)
is used as an index.
[0060] 18. A method for determining or evaluating an extract
isolated from inflamed tissue inoculated with vaccinia virus
wherein a promoting activity on the synthesis of collagen in
cultured intervertebral disc cells (bovine nucleus pulposus cells
or anulus fibrosus cells) is used as an index.
[0061] 19. An extract isolated from inflamed tissue inoculated with
vaccinia virus having a promoting activity on the synthesis of
proteoglycan in intervertebral disc cells (bovine nucleus pulposus
cells).
[0062] 20. An extract isolated from inflamed tissue inoculated with
vaccinia virus having a promoting activity of collagen synthesis in
intervertebral disc cells (bovine nucleus pulposus cells or anulus
fibrosus cells).
[0063] 21. A method for regenerating an intervertebral disc cell
extracellular matrix, comprising administering to a patient in need
of such treatment an extract isolated from inflamed tissue
inoculated with vaccinia virus.
[0064] 22. An agent for regenerating an intervertebral disc cell
extracellular matrix containing an extract isolated from inflamed
tissue inoculated with vaccinia virus.
[0065] 23. Use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for regenerating an intervertebral
disc cell extracellular matrix.
[0066] 24. A method for determining or evaluating an extract
isolated from inflamed tissue inoculated with vaccinia virus
wherein a regenerating activity of an intervertebral disc cell
extracellular matrix is used as an index.
[0067] 25. An extract isolated from inflamed tissue inoculated with
vaccinia virus having a regenerating activity of an intervertebral
disc cell extracellular matrix.
[0068] 26. A method for treating or preventing degenerative
intervertebral discs, comprising administering to a patient in need
of such treatment an extract isolated from inflamed tissue
inoculated with vaccinia virus.
[0069] 27. An agent for treating or preventing degenerative
intervertebral discs containing an extract isolated from inflamed
tissue inoculated with vaccinia virus.
[0070] 28. Use of an extract isolated from inflamed tissue
inoculated with vaccinia virus for treating or preventing
degenerative intervertebral discs.
[0071] 29. A method for treating or preventing a pain caused by
intervertebral disc degeneration, wherein an intervertebral disc
cell extracellular matrix is regenerated by administering to a
patient in need of such treatment an extract isolated from inflamed
tissue inoculated with vaccinia virus.
[0072] 30. A method for treating or preventing a pain caused by
intervertebral disc degeneration, wherein the synthesis of
proteoglycan in intervertebral disc cells (bovine nucleus pulposus
cells) is promoted by administering to a patient in need of such
treatment an extract isolated from inflamed tissue inoculated with
vaccinia virus.
[0073] 31. A method for treating or preventing a pain caused by
intervertebral disc degeneration, wherein the synthesis of collagen
in intervertebral disc cells (bovine nucleus pulposus cells or
anulus fibrosus cells) is promoted by administering to a patient in
need of such treatment an extract isolated from inflamed tissue
inoculated with vaccinia virus.
[0074] 32. The embodiments as described above in any one of
subparagraphs 1 to 31 wherein the inflamed tissue is a rabbit skin
tissue.
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