[go: up one dir, main page]

US20130164383A1 - Method for promoting the synthesis of collagen and proteoglycan in chondrocytes - Google Patents

Method for promoting the synthesis of collagen and proteoglycan in chondrocytes Download PDF

Info

Publication number
US20130164383A1
US20130164383A1 US13/821,617 US201113821617A US2013164383A1 US 20130164383 A1 US20130164383 A1 US 20130164383A1 US 201113821617 A US201113821617 A US 201113821617A US 2013164383 A1 US2013164383 A1 US 2013164383A1
Authority
US
United States
Prior art keywords
cells
intervertebral disc
vaccinia virus
synthesis
inflamed tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/821,617
Other versions
US20140255507A9 (en
Inventor
Koichi Masuda
Mitsuru Naiki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nippon Zoki Pharmaceutical Co Ltd
University of California San Diego UCSD
Original Assignee
Nippon Zoki Pharmaceutical Co Ltd
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nippon Zoki Pharmaceutical Co Ltd, University of California San Diego UCSD filed Critical Nippon Zoki Pharmaceutical Co Ltd
Priority to US13/821,617 priority Critical patent/US20140255507A9/en
Publication of US20130164383A1 publication Critical patent/US20130164383A1/en
Publication of US20140255507A9 publication Critical patent/US20140255507A9/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/065—Poxviridae, e.g. avipoxvirus
    • G01N2333/07—Vaccinia virus; Variola virus

Definitions

  • 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.
  • 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
  • NTP Neurotropin
  • NTP has been widely used to treat neuropathic pain, including post-herpetic neuralgia and low back pain in Japan.
  • 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.
  • NTP tumor necrosis factor- ⁇ (TNF- ⁇ ) 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.
  • ECM intervertebral disc cell extracellular matrix
  • 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.
  • chondrocytes such as intervertebral disc cells, such as bovine nucleus pulposus cells or annulus fibrosus cells, articular chondrocytes and meniscal cells.
  • 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.
  • a method for regenerating a chondrocyte extracellular matrix comprising administering to a patient in need of such treatment an extract isolated from inflamed tissue
  • 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.
  • AF anulus fibrosus cells
  • 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.
  • NP bovine nucleus pulposus cells
  • 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.
  • AF anulus fibrosus cells
  • 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.
  • NP bovine nucleus pulposus cells
  • 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.
  • PG proteoglycan
  • NP bovine nucleus pulposus
  • AF anulus fibrosus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the extract from inflamed tissue inoculated with vaccinia virus is produced, for example, according to the following process.
  • 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.
  • 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.
  • the inflamed tissues are collected and crushed, and 1 to 5 volumes of extraction solvent is added to make an emulsified suspension.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an acid such as hydrochloric acid, sulfuric acid or hydrobromic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • sodium chloride may be added to prepare a solution isotonic to physiological saline.
  • 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.
  • 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.
  • 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 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 50 value is 100 mg/kg of the preparation).
  • the mixture was adjusted to pH 10 with sodium hydroxide, stirred at 60° 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° 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.
  • 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.
  • 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 3 H-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.
  • the oxygen concentration and treatment with NTP did not affect NP and AF cell proliferation at both the 7 and 14 days time points.
  • PG synthesis was significantly increased with NTP treatment under culture at 5% O 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.
  • Collagen synthesis by NP cells in under 5% O 2 and NP and AF cells under 21% O 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 2 , +50%; NP, 21% O 2 , +63%; AF, 21% O 2 +47%).
  • 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.
  • 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.
  • chondrocytes such as intervertebral disc cells, articular chondrocytes and meniscal cells.
  • 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.
  • An agent for promoting the synthesis of proteoglycan and/or collagen in chondrocytes containing an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • 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.
  • a method for promoting the synthesis of proteoglycan in intervertebral disc cells comprising administering to a patient in need of such treatment an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • a method for promoting the synthesis of collagen in intervertebral disc cells comprising administering to a patient in need of such treatment an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • An agent for promoting the synthesis of proteoglycan in intervertebral disc cells containing an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • intervertebral disc cells bovine nucleus pulposus cells or anulus fibrosus cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Rheumatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)

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.

Description

    FIELD OF THE INVENTION
  • 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
  • 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.
  • The suppressive effect of NTP on tumor necrosis factor-α(TNF-α) 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
  • 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
  • The present invention is further illustrated by the accompanying drawings wherein:
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.)
  • 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.
  • 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.
  • 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.
  • The extract from inflamed tissue inoculated with vaccinia virus is produced, for example, according to the following process.
  • (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).
  • (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.
  • (c) The obtained filtrate or supernatant is made acidic and adsorbed onto an adsorbent such as activated carbon or kaolin.
  • (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ED50 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 ED50 value is 100 mg/kg of the preparation).
  • 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 ° C., and all pressures are atmospheric unless indicated to the contrary:
    • EXAMPLE 1
  • 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° C. for 30 minutes. After deproteinization by filtration, the filtrate was adjusted to pH 9 with sodium hydroxide, further heated at 90 to 100° 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° 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° 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
  • 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° C. and filtered. The filtrate was adjusted to weak alkalinity (pH 8.5 to 10.0) with sodium hydroxide, further heated at 100° 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
  • 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.
  • 1. Materials and Methods
  • 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 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.
  • 2. Results
  • 1) Cell Proliferation
  • The oxygen concentration and treatment with NTP did not affect NP and AF cell proliferation at both the 7 and 14 days time points.
  • 2) PG Synthesis
  • In NP cells, PG synthesis was significantly increased with NTP treatment under culture at 5% O2 (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.
  • 3) Collagen Synthesis
  • Collagen synthesis by NP cells in under 5% O2 and NP and AF cells under 21% O2 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% O2, +50%; NP, 21% O2, +63%; AF, 21% O2 +47%).
  • 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% O2 than under 21% O2 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% O2 and 21% O2, stimulation in the AF was only observed in the 21% O2 condition. Unlike the increased synthesis of PGs by NP cells in response to NTP, AF cells were more responsive to NTP under the 21% O2 condition. Because oxygen tension is significantly higher in the AF than in the NP, the greater response of AF cells to NTP at 21% O2 may also reflect optimal culture conditions.
  • 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
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 7. An agent for regenerating a chondrocyte extracellular matrix containing an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • 8. Use of an extract isolated from inflamed tissue inoculated with vaccinia virus for regenerating a chondrocyte extracellular matrix.
  • 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.
  • 10. An extract isolated from inflamed tissue inoculated with vaccinia virus having a regenerating activity of a chondrocyte extracellular matrix.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 22. An agent for regenerating an intervertebral disc cell extracellular matrix containing an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • 23. Use of an extract isolated from inflamed tissue inoculated with vaccinia virus for regenerating an intervertebral disc cell extracellular matrix.
  • 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.
  • 25. An extract isolated from inflamed tissue inoculated with vaccinia virus having a regenerating activity of an intervertebral disc cell extracellular matrix.
  • 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.
  • 27. An agent for treating or preventing degenerative intervertebral discs containing an extract isolated from inflamed tissue inoculated with vaccinia virus.
  • 28. Use of an extract isolated from inflamed tissue inoculated with vaccinia virus for treating or preventing degenerative intervertebral discs.
  • 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.
  • 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.
  • 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.
  • 32. The embodiments as described above in any one of subparagraphs 1 to 31 wherein the inflamed tissue is a rabbit skin tissue.

Claims (26)

What is claimed is:
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.
US13/821,617 2010-10-14 2011-10-11 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes Abandoned US20140255507A9 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/821,617 US20140255507A9 (en) 2010-10-14 2011-10-11 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US39319010P 2010-10-14 2010-10-14
US61393190 2010-10-14
US13/821,617 US20140255507A9 (en) 2010-10-14 2011-10-11 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes
PCT/US2011/055757 WO2012051173A2 (en) 2010-10-14 2011-10-11 A method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/055757 A-371-Of-International WO2012051173A2 (en) 2010-10-14 2011-10-11 A method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/858,396 Division US10711292B2 (en) 2010-10-14 2015-09-18 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Publications (2)

Publication Number Publication Date
US20130164383A1 true US20130164383A1 (en) 2013-06-27
US20140255507A9 US20140255507A9 (en) 2014-09-11

Family

ID=45938923

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/821,617 Abandoned US20140255507A9 (en) 2010-10-14 2011-10-11 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes
US14/858,396 Active 2034-11-29 US10711292B2 (en) 2010-10-14 2015-09-18 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/858,396 Active 2034-11-29 US10711292B2 (en) 2010-10-14 2015-09-18 Method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Country Status (8)

Country Link
US (2) US20140255507A9 (en)
EP (1) EP2627343B1 (en)
JP (1) JP5937089B2 (en)
KR (1) KR101894754B1 (en)
CN (1) CN103476923B (en)
AU (1) AU2011316756B2 (en)
CA (1) CA2808927C (en)
WO (1) WO2012051173A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220079995A1 (en) * 2018-02-20 2022-03-17 Tokai University Educational System Agent for promoting expression of n-acetylgalactosaminyltransferase containing extract from inflamed tissues inoculated with vaccinia virus

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI483729B (en) 2010-03-11 2015-05-11 Nippon Zoki Pharmaceutical Co Improving or therapeutic agent for chronic prostatitis, interstitial cystitis and/or urinary disturbance
CN103476923B (en) 2010-10-14 2015-04-29 日本脏器制药株式会社 A method for promoting the synthesis of collagen and proteoglycan in chondrocytes
TWI687223B (en) 2013-04-30 2020-03-11 日商日本臟器製藥股份有限公司 Extract and preparations containing said extract
US20170072321A1 (en) * 2013-05-22 2017-03-16 David S. Thompson Highly interactive fantasy sports interleaver
AU2016271772A1 (en) * 2015-05-29 2018-01-25 Nippon Zoki Pharmaceutical Co., Ltd. Pluripotent stem cell migration promoter
JP6966763B2 (en) * 2015-06-30 2021-11-17 日本臓器製薬株式会社 Improvement or treatment of chondromalacia patella
TWI705816B (en) * 2016-01-29 2020-10-01 國立大學法人千葉大學 Use of extract from inflamed tissue inoculated with vaccinia virus for manufacturing prevention or therapeutic agent or repair accelerator of muscle damage
JP7125732B2 (en) * 2016-09-09 2022-08-25 日本臓器製薬株式会社 Agent for improving or treating neurogenic bladder
US11207354B2 (en) 2016-09-23 2021-12-28 Osaka University Schwann cell differentiation promoting agent and a peripheral nerve regeneration promoting agent
JP2019142853A (en) * 2018-02-20 2019-08-29 学校法人東海大学 Agent for promoting expression of n-acetylgalactosaminyltransferase containing extract from inflamed tissues inoculated with vaccinia virus
CN111956669A (en) * 2020-09-07 2020-11-20 威世药业（如皋）有限公司 Preparation process of cowpox vaccine inflammation-causing rabbit fur extract injection

Family Cites Families (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53101515A (en) 1977-02-17 1978-09-05 Nippon Zoki Pharmaceutical Co Medicine having anodyne * sedative and antiallergic activity and production thereof
JPS5587724A (en) 1978-12-27 1980-07-02 Nippon Zoki Pharmaceut Co Ltd Selective immune enhancer
JP2651674B2 (en) 1987-07-23 1997-09-10 日本臓器製薬 株式会社 New physiologically active substance and method for producing the same
US4985254A (en) 1987-11-06 1991-01-15 Nippon Zoki Pharmaceutical Co., Ltd. Method of treating ischemic diseases
JPH0825885B2 (en) 1988-04-15 1996-03-13 日本臓器製薬株式会社 Idiopathic thrombocytopenic purpura treatment
JP2539665B2 (en) 1988-06-20 1996-10-02 日本臓器製薬株式会社 Neurological drug
JP2539669B2 (en) 1988-07-15 1996-10-02 日本臓器製薬株式会社 Diabetic neuropathy treatment
JP2588109B2 (en) 1993-03-19 1997-03-05 日本臓器製薬株式会社 Painkillers
JP2594222B2 (en) 1993-09-28 1997-03-26 日本臓器製薬株式会社 New physiologically active substance-KF
JP3852786B2 (en) 1995-04-25 2006-12-06 日本臓器製薬株式会社 Bone atrophy improving agent
JP4033936B2 (en) 1997-01-08 2008-01-16 日本臓器製薬株式会社 Nitric oxide production inhibitor
JPH1180005A (en) * 1997-09-12 1999-03-23 Nippon Zoki Pharmaceut Co Ltd Therapeutic agent for osteoporosis
JPH11139977A (en) 1997-11-07 1999-05-25 Nippon Zoki Pharmaceut Co Ltd Nef action suppressant
JP2000016942A (en) 1998-04-27 2000-01-18 Nippon Zoki Pharmaceut Co Ltd Therapeutic agent for ischemic disease
CN100425692C (en) 1998-06-02 2008-10-15 株式会社富吉摩托普拉泽兹 Method for preparing rabbitpox culturing tissue containing high-quality physiological activity
CN1055249C (en) 1998-07-15 2000-08-09 沈继平 Analgesia medicine and its mfg. method
JP2000143536A (en) 1998-11-13 2000-05-23 Nippon Zoki Pharmaceut Co Ltd Antihydropic agent
AU754989B2 (en) 1998-11-16 2002-11-28 Nippon Zoki Pharmaceutical Co., Ltd. A therapeutic agent for intractable vasculitis
JP2000336034A (en) 1999-03-19 2000-12-05 Nippon Zoki Pharmaceut Co Ltd Promoter for chemokine production
CN1272365A (en) 1999-04-15 2000-11-08 日本脏器制药株式会社 Physiological active material
US6723335B1 (en) * 2000-04-07 2004-04-20 Jeffrey William Moehlenbruck Methods and compositions for treating intervertebral disc degeneration
TWI243055B (en) 2000-04-13 2005-11-11 Nippon Zoki Pharmaceutical Co Pharmaceutical composition for use in treatment of dermatitis
JP4711523B2 (en) 2001-02-13 2011-06-29 日本臓器製薬株式会社 Hypoalbuminemia improving agent
TWI342778B (en) 2002-10-31 2011-06-01 Nippon Zoki Pharmaceutical Co Therapeutic agent for fibromyalgia
US7727550B2 (en) * 2003-02-21 2010-06-01 The Uab Research Foundation Biologically active native biomatrix composition
JP2004300146A (en) 2003-03-17 2004-10-28 Nippon Zoki Pharmaceut Co Ltd Preventive agent against infectious disease
KR101307999B1 (en) 2004-12-01 2013-09-12 니폰 조키 세야쿠 가부시키가이샤 Dried material and method for the manufacture thererof
US20060134646A1 (en) 2004-12-17 2006-06-22 Ansari Aftab A Method for treatment of HIV infection
TWI406664B (en) 2006-03-30 2013-09-01 Univ Kyoto Agent increasing the production of thioredoxin
CN101528947A (en) * 2006-09-06 2009-09-09 日本脏器制药株式会社 Method for study, determination or evaluation by gene expression analysis
JPWO2009028605A1 (en) 2007-08-31 2010-12-02 国立大学法人九州大学 Preventive or alleviating peripheral neuropathy with anticancer agents
TWI483729B (en) 2010-03-11 2015-05-11 Nippon Zoki Pharmaceutical Co Improving or therapeutic agent for chronic prostatitis, interstitial cystitis and/or urinary disturbance
CN103476923B (en) 2010-10-14 2015-04-29 日本脏器制药株式会社 A method for promoting the synthesis of collagen and proteoglycan in chondrocytes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220079995A1 (en) * 2018-02-20 2022-03-17 Tokai University Educational System Agent for promoting expression of n-acetylgalactosaminyltransferase containing extract from inflamed tissues inoculated with vaccinia virus

Also Published As

Publication number Publication date
CN103476923B (en) 2015-04-29
CN103476923A (en) 2013-12-25
WO2012051173A3 (en) 2013-09-26
JP5937089B2 (en) 2016-06-22
US20140255507A9 (en) 2014-09-11
WO2012051173A2 (en) 2012-04-19
KR20140020225A (en) 2014-02-18
KR101894754B1 (en) 2018-09-04
AU2011316756B2 (en) 2015-01-22
EP2627343A2 (en) 2013-08-21
CA2808927A1 (en) 2012-04-19
EP2627343B1 (en) 2018-01-10
US20160010135A1 (en) 2016-01-14
CA2808927C (en) 2018-10-30
EP2627343A4 (en) 2014-08-06
US10711292B2 (en) 2020-07-14
JP2013544785A (en) 2013-12-19
AU2011316756A1 (en) 2013-03-14

Similar Documents

Publication Publication Date Title
US10711292B2 (en) Method for promoting the synthesis of collagen and proteoglycan in chondrocytes
AU2008292407B2 (en) Prophylactic or alleviating agent for peripheral nerve disorder induced by anti-cancer agent
CA2792085A1 (en) Ameliorating or therapeutic agent comprising an extract from inflamed tissue inoculated with vaccinia virus for chronic prostatitis, interstitial cystitis and/or urination disorders
US11129976B2 (en) Test method
US20240408237A1 (en) Method and pharmaceutical composition for treating a cartilage damage using sox9 gene
US11207354B2 (en) Schwann cell differentiation promoting agent and a peripheral nerve regeneration promoting agent
KR102526447B1 (en) A composition for preventing or treating of liver disease comprising conditioned medium of tonsil-derived mesenchymal stem cell
CN110179828B (en) N-acetylgalactosamine transferase expression promoter containing vaccinia virus inoculated inflammatory tissue extract
JP6849191B2 (en) Preventive or therapeutic agents for muscle damage
JP2017014204A (en) Improvement or treatment agent of chondromalacia patella
Sathiyapriya et al. HEPATOPROTECTIVE EFFECT OF Ipomoea mauritiana jacq AGAINST AMOXICILLIN INDUCED TOXICITY IN ALBINO RATS.
HK1143734B (en) Prophylactic or alleviating agent for peripheral nerve disorder induced by anti-cancer agent

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MASUDA, KOICHI;REEL/FRAME:027138/0857

Effective date: 20111007

Owner name: NIPPON ZOKI PHARMACEUTICAL CO., LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NAIKI, MITSURU;REEL/FRAME:027138/0992

Effective date: 20111012

AS Assignment

Owner name: NIPPON ZOKI PHARMACEUTICAL CO., LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NAIKI, MITSURU;REEL/FRAME:030059/0645

Effective date: 20111012

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MASUDA, KOICHI;REEL/FRAME:030059/0471

Effective date: 20111007

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION