JP2005247836A - Pharmaceutical composition for treating pain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for treating pains, especially treatment-resistant pain such as cancerous pain, neurogenic pain and inflammatory pains by studying actions of oncostatin M on a pain-responsive neuron. <P>SOLUTION: The composition comprises an oncostatin M antagonist or the oncostatin M. Furthermore, the composition comprises a transgenic vector containing a nucleic acid in which a cytotoxic gene is linked to a promoter of an oncostatin M receptor β-chain gene. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to the field of drugs for treating pain.

(Oncostatin M and its receptor)
Oncostatin M was discovered in the late 1980s as a factor that suppresses the growth of human A375 melanoma cells, and was named as an inhibitor of cancer. Isolation of the human oncostatin M gene reveals that oncostatin M belongs to the interleukin 6 (IL-6) family and is similar in activity and structure particularly to leukemia inhibitory factor (LIF). It was. However, since then, the gene encoding mouse oncostatin M has not been identified for a long time, and the understanding has not progressed beyond that of LIF-like cytokines. Thereafter, as a result of analysis of the signal transduction mechanism of cytokine, as a result of identification of a gene controlled by transcription factor STAT5 activated by cytokine stimulation, cDNA of mouse oncostatin M was cloned (SEQ ID NO: 4), and oncostatin M Analysis progressed. There was a STAT5 binding sequence in the promoter region of the oncostatin M gene, and it was shown that the expression of the oncostatin M gene was induced via STAT5 by cytokine stimulation. Oncostatin M has a wide variety of physiological activities and is known to be involved in embryogenesis, inflammatory reaction, hematopoiesis, and liver development. Depending on the cell type, it exhibits activities such as growth promotion, growth inhibition, and differentiation induction. .

  The oncostatin M receptor is a heterodimer of the gp130 molecule and the oncostatin M receptor β chain (OSMRβ). In contrast, the receptor for LIF belonging to the IL-6 family, like Oncostatin M, is a heterodimer of the gp130 molecule and the leukemia inhibitory receptor β chain (LIFRβ). These β chains are essential for ligand binding and signal transduction, and have an intracellular domain with a length corresponding to the gp130 molecule.

  In recent years, Oncostatin M specific biological activity (eg, proliferation of pluripotent hematopoietic progenitor cells and formation of endothelial cell clusters, hepatocyte differentiation, and neonatal Sertoli cell proliferation) has been It has been reported to mediate chains.

  We have previously shown that Oncostatin M receptor β chain is expressed during embryogenesis and in various organs including the nervous system within adult mice. (Tamura et al., Mech Dev 115: 127-131 (2002); Tamura et al., Neuroscience 119 (2003) 991-997; Tamura et al., Eur. J. Neurosci 17: 2287-2298 (2003)). In the spinal ganglia (DRG), the oncostatin M receptor β chain has been shown to be expressed in peptidergic nociceptive neurons. This peptidergic nociceptive neuron expressed both vanilloid receptor (VR1) and P2X3 purinergic receptors. (Tamura et al., EJ Neurosci, 17, 2287-2298 (2003)). However, the association between oncostatin M receptor β chain and pain is not taught or suggested.

(Neuropathic pain)
Pain is broadly divided into acute pain, inflammatory pain, and neuropathic pain. Among them, neuropathic pain is a chronic disease that is very refractory. As non-steroidal anti-inflammatory drugs (NSAIDs) and narcotics (opioids) are currently used as analgesics, neuropathic pain shows resistance to these drugs. Allodynia in neural pain (a phenomenon of pain induced by tactile stimulation) is persistent, and maintenance of allodynia in the spinal cord includes glutamate receptors (particularly NMDA receptors), glutamate transporters, NO (nitrogen monoxide), and Prostaglandins (particularly PGE ₂ and PGF _2α ) are thought to be involved (Toshiaki Minami, Yumi Doi, Tadatoshi Muratani, Wataru Nishimura, Mikio Nishizawa, Seiji Ito: Role of prostaglandins in the spinal cord for pain transmission Edited by Nobuhiko Ogata and Ryusuke Kashiwagi, “Pain Basics and Clinics, Shinko Trading Medical Publishing Department, pp. 138-149, 2003).

  As a nerve fiber associated with a hypersensitivity reaction in neuropathic pain, a type III nerve fiber has been proposed as a nerve fiber insensitive to capsaicin, glutamate as a transmitter, and a prostaglandin I2 agonist or the like (Ueda). Hiroshi, Makoto Inoue: Morphine Tolerance and Morphine Insensitivity Neuropathic Pain, Nobuhiko Ogata, Ryusuke Kashiwagi, “Pain Basics and Clinical, Shinko Trader Publishing Department”, 152-165, 2003), Neuropathic pain Drugs that do not exhibit resistance for the treatment have not been developed yet. In addition, the development of drugs for treating pain other than neuropathic pain (for example, acute pain, inflammatory pain, cancer pain) is still insufficient. For example, morphine is the first choice for cancer pain and works well in the early days, but it disappears after a while (tolerance) and exhibits central nervous symptoms such as hallucinations. Therefore, development of an effective drug for various pains is desired.

Pain is transmitted through the spinal ganglia (DRG). Therefore, for example, it is considered that treatment of pain is possible by removing nerves passing through DRG or blocking signals. However, it is difficult to remove only nerves physically involved in pain. On the other hand, since IB4 is a marker that is expressed in 40% of nerves of DRG, a method for alleviating pain sensation by specifically removing IB4-expressing neurons has been proposed (Non-patent Document 6). However, DRG neurons include c-ret ⁺ neurons and trkA ⁺ neurons, but IB4 is expressed exclusively in c-ret ⁺ neurons, and 40% of all DRG neurons are IB4. ^Since it is ⁺ , using IB4 expression as an index may result in the removal of DRG neurons more than necessary, and since the cells to be removed are in a biased population of neurons, its side effects Is concerned.
Tamura et al., Mech Dev 115: 127-131 (2002). Tamura et al., Neuroscience 119 (2003) 991-997. Tamura et al. J. et al. Neurosci. 17, 2287-2298 (2003) Toshiaki Minami, Yumi Doi, Tadatoshi Muratani, Wataru Nishimura, Mikio Nishizawa, Seiji Ito: The role of prostaglandins in the spinal cord for pain transmission 138-149 pages, 2003 Hiroshi Ueda, Makoto Inoue: Morphine tolerance and morphine insensitivity neuropathic pain, Nobukuni Ogata, Ryusuke Takagi, “Pain Basics and Clinic, Shinko Trader Publishing Department”, 152-165 pages, 2003 Vulchanova et al., Neuroscience, Vol. 108, no. 1, pages 143-155, 2001.

  Thus, there is an increasing need in the art for drugs to treat pain, particularly resistant pain (eg, cancer pain, neuropathic pain, inflammatory pain, etc.). An object of the present invention is to meet such a demand.

  As a result of the present inventors diligently examining the above problems, the pain response is unexpectedly suppressed by inhibiting / suppressing a reaction triggered by oncostatin M in cells expressing the oncostatin M receptor. It was partially completed by finding out that it was done. Furthermore, surprisingly, the present inventors have found that the pain response is also suppressed by administering Oncostatin M to the peripheral nerve side of the central nervous system, and completed the present invention.

Accordingly, the present invention provides the following.
1. A pharmaceutical composition for treating pain comprising an oncostatin M antagonist and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to item 1, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant.
3. Item 3. The pharmaceutical composition according to Item 2, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody.
4). Item 4. The pharmaceutical composition according to Item 3, wherein the anti-oncostatin M receptor antibody forms a conjugate with a cytotoxin.
5). Item 5. The pharmaceutical composition according to Item 4, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase.
6). Item 2. The pharmaceutical composition according to Item 1, wherein the oncostatin M antagonist is contained so that the local concentration at the site of administration is 0.1 to 100 mg / ml.
7). The pharmaceutical composition according to item 1, further comprising an anti-oncostatin M antagonist antibody conjugated with a cytotoxin.
8). 2. The pharmaceutical composition according to item 1, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
9. Item 2. The pharmaceutical composition according to Item 1, further comprising at least one of a nonsteroidal anti-inflammatory analgesic and a narcotic.
10. Item 1. further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin A pharmaceutical composition as described.
11. Use of an oncostatin M antagonist in the manufacture of a medicament for treating pain.
12 12. Use according to item 11, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant.
13. 13. Use according to item 12, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody.
14 14. Use according to item 13, wherein the anti-oncostatin M receptor antibody forms a conjugate with a cytotoxin.
15. 15. Use according to item 14, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase.
16. Item 12. The use according to Item 11, which is contained in the medicament so that the local concentration at the site where the oncostatin M antagonist is administered is 0.1-100 mg / ml.
17. 12. Use according to item 11, further comprising the use of an anti-oncostatin M antagonist antibody conjugated to a cytotoxin.
18. 12. Use according to item 11, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
19. A kit for treating pain, comprising the pharmaceutical composition according to item 1.
20. Item 20. The kit of item 19, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant.
21. 21. A kit according to item 20, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody.
22. Item 22. The kit according to Item 21, wherein the anti-oncostatin M receptor antibody forms a conjugate with a cytotoxin.
23. The kit according to item 22, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase.
24. Item 20. The kit according to Item 19, which is contained in the pharmaceutical composition so that the local concentration at the site where the oncostatin M antagonist is administered is 0.1-100 mg / ml.
25. 20. A kit according to item 19, further comprising an anti-oncostatin M antagonist antibody conjugated with a cytotoxin.
26. 20. A kit according to item 19, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
27. Item 20. The kit according to Item 19, further comprising at least one of a nonsteroidal anti-inflammatory analgesic and a narcotic.
28. Item 27, further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin The described kit.
29. A pharmaceutical composition for treating pain, the composition comprising:
(A) a gene transfer vector comprising a control region of an oncostatin M receptor β chain gene and a cytotoxic gene operably linked to the control region; and (b) a pharmaceutically acceptable carrier. A pharmaceutical composition for treating pain, comprising:
30. 30. The pharmaceutical composition according to item 29, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene.
31. 31. The pharmaceutical composition according to item 30, wherein the promoter sequence is a sequence consisting of the sequence set forth in SEQ ID NO: 1.
32. 30. The pharmaceutical composition according to item 29, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus.
33. 33. The pharmaceutical composition according to item 32, wherein the gene transfer vector is derived from adenovirus, adeno-associated virus, retrovirus, or lentivirus.
34. 33. The pharmaceutical composition according to item 32, wherein the gene transfer vector is derived from a retrovirus.
35. 30. The pharmaceutical composition of item 29, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, ricin, and herpes thymidine kinase.
36. 30. The pharmaceutical composition according to item 29, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
37. 30. The pharmaceutical composition of item 29, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic.
38. Item 29, further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. A pharmaceutical composition as described.
39. Use of a gene transfer vector in the manufacture of a medicament for treating pain, the vector comprising a control region of an oncostatin M receptor β chain gene and a cytotoxic gene operably linked to the control region ,use.
40. 40. Use according to item 39, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene.
41. 41. Use according to item 40, wherein the sequence of the promoter is a sequence consisting of the sequence described in SEQ ID NO: 1.
42. 40. Use according to item 39, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus.
43. The use according to item 42, wherein the gene transfer vector is derived from adenovirus, adeno-associated virus, retrovirus, or lentivirus.
44. The use according to item 42, wherein the gene transfer vector is derived from a retrovirus.
45. 40. Use according to item 39, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, ricin, and herpes thymidine kinase.
46. 40. Use according to item 39, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
47. 30. A kit for treating pain, comprising the pharmaceutical composition according to item 29.
48. 48. The kit according to item 47, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene.
49. Item 49. The kit according to Item 48, wherein the promoter sequence is a sequence consisting of the sequence set forth in SEQ ID NO: 1.
50. 48. A kit according to item 47, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus, and lentivirus.
51. 51. A kit according to item 50, wherein the gene transfer vector is derived from an adenovirus, an adeno-associated virus, a retrovirus, or a lentivirus.
52. 51. The kit according to item 50, wherein the gene transfer vector is derived from a retrovirus.
53. 48. A kit according to item 47, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, lysine, and herpes thymidine kinase.
54. 48. A kit according to item 47, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
55. 48. The kit of item 47, further comprising at least one of a nonsteroidal anti-inflammatory analgesic and a narcotic.
56. Item 47, further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. The described kit.
57. A pharmaceutical composition for treating pain comprising at least one of oncostatin M or oncostatin M homolog and a pharmaceutically acceptable carrier,
Here, an oncostatin M homolog is a polypeptide that is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid set forth in SEQ ID NO: 4, 6 or 8, and has oncostatin M activity, wherein Oncostatin M activity is the activity of promoting the proliferation of pluripotent hematopoietic progenitor cells, the activity of promoting the formation of endothelial cell clusters, the activity of promoting differentiation of hepatocytes, the activity of promoting the proliferation of newborn Sertoli cells, and the induction of production of acute phase proteins from hepatocytes A pharmaceutical composition which is at least one activity selected from the group consisting of an activity, a TIMP1 expression-inducing activity, and a liver regeneration promoting activity.
58. 58. The pharmaceutical composition according to item 57, wherein Oncostatin M or Oncostatin M homolog is contained so that the local concentration at the site of administration is 0.1-100 mg / ml.
59. 58. The pharmaceutical composition of item 57, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
60. 58. The pharmaceutical composition of item 57, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic.
61. Item 57, further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin A pharmaceutical composition as described.
62. 58. The pharmaceutical composition of item 57, formulated to be suitable for subcutaneous or intramuscular administration.
63. 58. The pharmaceutical composition of item 57, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain.
64. 58. The pharmaceutical composition of item 57, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic.
65. Item 57, further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin A pharmaceutical composition as described.
66. Use of Oncostatin M or Oncostatin M homologue in the manufacture of a medicament for treating pain.
67. 58. A kit for treating pain, comprising the pharmaceutical composition according to item 57.
68. A pharmaceutical composition for treating pain, the composition comprising:
(A) a gene transfer vector comprising an oncostatin M gene or an oncostatin M homolog gene,
Here, an oncostatin M homolog is a polypeptide that is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid set forth in SEQ ID NO: 4, 6 or 8, and has oncostatin M activity, wherein Oncostatin M activity is the activity of promoting the proliferation of pluripotent hematopoietic progenitor cells, the activity of promoting the formation of endothelial cell clusters, the activity of promoting differentiation of hepatocytes, the activity of promoting the proliferation of newborn Sertoli cells, and the induction of production of acute phase proteins from hepatocytes A gene transfer vector which is at least one activity selected from the group consisting of activity, TIMP1 expression-inducing activity, and liver regeneration promoting activity,
And (b) a pharmaceutical composition for treating pain comprising a pharmaceutically acceptable carrier.
69. 70. The pharmaceutical composition according to item 68, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus.
70. 70. The pharmaceutical composition according to item 69, wherein the gene transfer vector is derived from an adenovirus, an adeno-associated virus, a retrovirus, or a lentivirus.
71. 71. The pharmaceutical composition according to item 70, wherein the gene transfer vector is derived from a retrovirus.

  The pharmaceutical composition of the present invention has an effect that it can treat pain, particularly resistant pain (for example, cancer pain, neuropathic pain, inflammatory pain, etc.).

  The present invention will be described below. Throughout this specification it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “Oncostatin M” refers to a mouse protein encoded by the nucleic acid sequence of SEQ ID NO: 4, a human protein encoded by the nucleic acid sequence of SEQ ID NO: 6, unless otherwise specified, SEQ ID NO: It refers to the rat protein encoded by the eight nucleic acid sequences, and homologs and orthologs of that protein, as well as all variants and allelic variants of these proteins, homologs and orthologs.

  As used herein, an “oncostatin M homolog” is a protein encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid set forth in SEQ ID NO: 4, 6, or 8, for example, Refers to a polypeptide having at least one oncostatin M activity, or has an amino acid sequence in which one or several amino acid residues have been deleted, added, substituted in the amino acid sequence of natural oncostatin M, and at least A polypeptide having one oncostatin M activity. Oncostatin M activity includes pluripotent hematopoietic progenitor cell proliferation promoting activity, endothelial cell cluster formation promoting activity, hepatocyte differentiation promoting activity, neonatal Sertoli cell growth promoting activity, acute phase protein production from hepatocytes Inducing activity, TIMP1 expression inducing activity, liver regeneration promoting activity, growth inhibition activity of normal breast epithelial cells and normal breast epithelial cells, growth promoting activity of AIDS-related Kaposi sarcoma cells and myeloma cells and plasmacytoma cells, mitogenic activity Activity to stimulate cutaneous fibroblasts via activated protein kinase (MAPK) -dependent pathway, proliferation-stimulating activity of endothelium-like cells in primary cultures of AGM-derived cells, acute phase protein (APP) in hepatocellular carcinoma and hepatocytes ) Activity to stimulate synthesis, activity to stimulate IL-6 production in cultured endothelial cells Activity to stimulate IL-6 receptor production in HepG2 cells, IL-1 and GM-CSF IL-1 induced expression in synovial fibroblasts and lung fibroblasts, chemokine proliferation in human endothelial cells Induced activity of related cytokines α and β mRNA, long-term expression-inducing activity of P-selectin and E-selectin in human endothelial cells, activity of inducing the expression of intercellular adhesion molecule (ICAM-1) and VCAM-1 by human endothelial cells, human lung cells And activity to increase TMIP-1 expression in fibroblasts of synovial origin, activity to increase TMIP-1 expression in cultured human synovial lining cells, IL-1β-inducible TMIP- in cultured human synovial lining cells 3 Inhibits expression, enhances MMP-1 and MMP-3 expression in astrocytes Sex, activity to enhance MMP-1 and MMP-9 expression in fibroblasts, activity to stimulate TIMP-1 mRNA in NIH-3T3 cells, activity to stimulate α1-antichymotrypsin synthesis in lung-derived epithelial cells, Activity to upregulate α1-antichymotrypsin levels in lung-derived epithelial cells, activity to upregulate low density lipoprotein receptor and protein S expression in HepG2 cells, CYP1A2 and CYP3A4 (cytochromes) at the transcriptional level in human hepatocytes Activity to down-regulate P450 isozyme), increase collagen production in hepatic stellate cells without inducing collagen mRNA, enhance E-cadherin-based cell adhesion formation in hepatocytes via K-Ras Activity, via STAT3 Activity of down-regulating D1 cyclin and D2 cyclin expression in fetal liver parenchymal cells, activity of up-regulating D cyclin expression in mature liver parenchymal cells of regenerated liver, expression of CD45 positive hematopoietic cells in fetal liver Examples include, but are not limited to, inducing activity, proliferation-stimulating activity of endothelial-like cells in primary cultures of mouse AGM cells, and activity inducing a myeloproliferative phenotype.

As used herein, “stringent conditions” for hybridization are those hybridization conditions that correspond to the highest T _m values (eg, 50% formamide, 5 × or 6 × SSC). Methods for determining the _Tm of nucleic acid sequences are well known.

  As used herein, an “antagonist” is an antagonist that acts to antagonize the binding of a biological agent (eg, a ligand such as Oncostatin M) to a receptor, and is itself physiologically mediated through that receptor. A factor that does not show an action. Antagonists, blockers (blockers), inhibitors (inhibitors) and the like are also encompassed by this antagonist. Anti-oncostatin M receptor antibodies, anti-oncostatin M receptor antibodies can also act as antagonists of oncostatin M.

  As used herein, an “oncostatin M antagonist” refers to a substance that acts antagonistically to the oncostatin M binding to the oncostatin M receptor, but does not itself act like oncostatin M. . Oncostatin M antagonists include, but are not limited to, various well-known substances listed below: anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, oncostatin M receptor A fusion protein comprising at least a portion, an anti-gp130 antibody, and an oncostatin M variant.

As used herein, “antibody” refers to antibodies or antibody fragments including polyclonal and monoclonal antibodies, antibody fragments, single chain antibodies, human and chimeric antibodies, and fused to phage coat or cell surface proteins , And other antibodies described herein that are known in the art, refer to a polypeptide that specifically binds an antigen. The antibodies herein also include chemically modified antibodies (eg, glycosylation, etc.) and antibodies fused to other proteins such as toxins (eg, diphtheria toxin, and ricin). The antibodies of the present invention can exhibit a specific binding affinity for an antigen of at least about 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ M ⁻¹ and can be polyclonal, monoclonal, recombinant or other It can be a product. Polypeptides that can be used as antigens to produce the antibodies of the present invention include Oncostatin M, Oncostatin M receptor, Oncostatin M receptor, or gp130, or variants thereof, or fragments of these polypeptides. However, it is not limited to these.

  In the present invention, various commercially available antibodies can be used. For example, anti-mouse oncostatin M antibody (R &D; catalog number AF-495-NA), or anti-human oncostatin M antibody (R &D; catalog number AF-295-NA or MAB295), or anti-mouse oncostatin Use of M receptor β antibody (R &D; catalog number AF662) or anti-mouse oncostatin M receptor β / Fc chimeric antibody (R &D; catalog number 662-OR-100) as the oncostatin M antagonist of the present invention. Can do. Moreover, the antibody which can be used as the oncostatin M antagonist of this invention can be prepared using the various well-known technique described below.

  For antibody production, a host such as a goat, sheep, cow, guinea pig, rabbit, rat or mouse is immunized by injection with an antigen protein or any site, fragment or oligopeptide that retains immunogenic properties. obtain. In selecting an antigen polypeptide for antibody induction, it is not necessary to retain biological activity; however, a protein fragment or oligopeptide must be immunogenic and preferably antigenic. Immunogenicity can be measured by injecting the polypeptide and adjuvant into animals (eg, rabbits) and assaying for the appearance of antibodies directed against the injected polypeptide (eg, Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York (1988), incorporated herein by reference in its entirety for all purposes). Peptides used to induce specific antibodies typically have an amino acid sequence consisting of at least 5 amino acids, preferably at least 8 amino acids, more preferably at least 10 amino acids. Usually, they mimic all or adjacent portions of the amino acid sequence of the native protein or have substantial sequence identity with it. A short region of antigenic protein amino acids can be fused to regions of another protein such as keyhole limpet hemocyanin and antibodies raised against the chimeric molecule. Depending on the host species, various adjuvants can be used to increase the immunological response.

  The antigen is presented to the immune system in a manner determined by a method appropriate for the animal. These and other parameters are generally well known to immunologists. Typically, injections are administered intramuscularly, intramuscularly, intradermally, around lymph nodes or intraperitoneally. Routine methods involving affinity purification can precipitate, isolate and purify the immunoglobulin produced by the host.

  In accordance with the present invention, an anti-oncostatin M receptor antibody conjugated with a cytotoxin can be used to specifically remove cells expressing the oncostatin M receptor. Alternatively, in accordance with the present invention, a secondary antibody that binds to an anti-oncostatin M receptor antibody and that is conjugated to a cytotoxin is used to specifically remove cells that express the oncostatin M receptor. Can do. Such a secondary antibody is, for example, an antibody that specifically recognizes the Fc portion of a rat antibody when the anti-oncostatin M receptor antibody is a rat-derived antibody. For example, an antibody conjugated with saporin, a cytotoxin, is commercially available (Funakoshi Corporation, Tokyo).

A) Monoclonal antibody:
Monoclonal antibodies against antigenic proteins and peptides can be prepared according to the methods of the invention using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include the hybridoma technology first described by Koehler and Milstein (Nature 256: 495 [1975]), the human B cell hybridoma technology (Kosbor et al., 1983, Immunol. Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA, 80: 2026), and EBV hybridoma technology (Cole et al., MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R Liss Inc., New York, NY, pp. 77-96 [1985]). Not.

  In certain embodiments, an appropriate animal is selected and followed by an appropriate immunization protocol. Production of non-human monoclonal antibodies (eg, mice, rabbits, horses) is well known and can be achieved, for example, by immunizing animals with a preparation containing the antigen or fragment thereof. In one method, after an appropriate time, the animal's spleen is excised and individual spleen cells are typically fused to immortal myeloma cells under appropriate selection conditions. The cells are then separated into clones and the supernatant of each clone (eg, hybridoma) is tested for the production of appropriate antibodies specific for the desired region of the antigen. Techniques for producing antibodies are well known in the art. See, eg, Goding et al., MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd edition) Acad. Press, NY and Harlow and Lane (supra), each of which is incorporated herein by reference in its entirety for all purposes. ) Other suitable techniques include in vitro exposure of lymphocytes to antigenic polypeptides or to selection of libraries of antibodies in phage or similar vectors.

B) Human antibody:
In another aspect of the invention, human antibodies against antigen polypeptides are provided. Using transgenic animals having elements of the human immune system (see, eg, US Pat. Nos. 5,569,825 and 5,545,806, both of which are incorporated herein by reference in their entirety for all purposes) Alternatively, human monoclonal antibodies against known antigens can also be made using human peripheral blood cells (Casali et al., 1986, Science 234: 476). Some human antibodies are selected by competitive binding experiments or to have the same epitope specificity as a particular mouse antibody.

  In another embodiment, the human antibody against the antigenic polypeptide is derived from human B cells according to the general protocol outlined by Huse et al., 1989, Science 246: 1275 (incorporated herein by reference). Can be produced by screening a DNA library. An antibody that binds to the antigen polypeptide is selected. The sequence encoding such an antibody (or binding fragment) is then cloned and amplified. The protocol described by Huse is often used with phage display technology.

C) Humanized or chimeric antibodies:
The present invention also provides antibodies that are chimeric, human-like or humanized to reduce their potential antigenicity without reducing their affinity for their target. The preparation of chimeric, human-like and humanized antibodies has been described in the art (eg, US Pat. Nos. 5,585,089 and 5,530,101; Queen et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 10029; And Verhoeyan et al., 1988, Science 239: 1534, all of which are incorporated herein by reference for all purposes.Humanized immunoglobulins are variable framework regions substantially derived from human immunoglobulins. (Referred to as acceptor immunoglobulin) and substantially non-human (eg, mouse) immunoglobulin derived complementarity determining regions (referred to as donor immunoglobulins), if present, the constant regions derived from substantially human immunoglobulins. is there.

  In some applications, such as administration to human patients, the humanized (as well as human) antibodies of the present invention provide several advantages over antibodies from murine or other species: (1) human The immune system should not recognize the framework or constant region of the humanized antibody as foreign, and thus the antibody response to such injected antibodies is totally foreign mouse antibodies or partial (2) Since the effector portion of the humanized antibody is human-derived, it may interact better with other parts of the human immune system; and ( 3) The injected humanized antibody has a half-life substantially equal to that of a naturally occurring human antibody, allowing smaller and less frequent doses than other species of antibodies. As indicated from the foregoing, pharmaceutical compositions comprising antibodies can be used to treat pain.

D) Phage display:
The present invention also includes phage display methods (eg, Dower et al., WO91 / 17271 and McCafferty et al., WO92 / 01047; and Vaughan et al., 1996, Nature Biotechnology, 14: 309; all of which are incorporated herein for all purposes. Which is incorporated by reference in its entirety). In these methods, a library of phage is produced. Here, the members present different antibodies on their outer surface. Antibodies are usually presented as Fv or Fab fragments. Phages displaying antibodies with the desired specificity are selected by their abundance of affinity for the antigen polypeptide.

In a variation of the phage display method, humanized antibodies with the binding specificity of the selected murine antibody can be produced. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (ie, murine starting material) and different heavy chain variable regions. The heavy chain variable region is obtained from a library of rearranged human heavy chain variable regions. Phages that exhibit strong specific binding to the antigen polypeptide (eg, at least 10 ⁸ M ⁻¹ and preferably at least 10 ⁹ M ⁻¹ ) are selected. This phage-derived human heavy chain variable region then serves as starting material for the construction of additional phage libraries. In this library, each phage displays the same heavy chain variable region (ie, the region identified from the original display library) and a different light chain variable region. The light chain variable region is obtained from a library of rearranged human variable light chain regions. Again, phages that exhibit strong specific binding are selected. These phage display the variable region of the complete antibody. These antibodies usually have the same or similar epitope specificity as the murine starting material.

E) Hybrid antibody:
The present invention also provides a hybrid antibody that shares the specificity of the antibody for the antigen polypeptide but can also specifically bind to the second site. In such hybrid antibodies, one heavy and light chain pair is usually derived from an antibody reactive to the first antigen, and the other pair is derived from an antibody reactive to the second antigen. This results in multifunctional properties, ie the ability to bind to at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing each component antibody or by recombinant techniques. Such hybrids can be used to deliver a compound (ie, drug) to a cell expressing Oncostatin M or Oncostatin M receptor (ie, a cytotoxic agent is specifically delivered).

  The immunoglobulins of the invention can also be fused to functional regions (eg, enzymes) from other genes to produce fusion proteins (eg, immunotoxins) having useful properties.

F) General:
The antibodies of the invention can be of any idiotype, such as IgM, IgD, IgG, IgA and IgE, with IgG, IgA and IgM often preferred. A humanized antibody may comprise sequences from more than one class or isotype.

In another embodiment of the invention, fragments of the intact antibodies described above are provided. Typically, these fragments can compete for specific binding to the antigenic polypeptide with the intact antibody from which they are derived, at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ M ⁻¹ , or 10 ¹⁰ It can bind with an affinity of M- ¹ . Antibody fragments comprise separate heavy chains, light chains, Fab, Fab ′, (ab ′) 2, Fabc, and Fv. Fragments can be generated by enzymatic or chemical separation of intact immunoglobulins. For example, F (ab ') 2 fragments can be obtained from IgG molecules by protein digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane (supra). it can. Fab fragments can be obtained from F (ab ') 2 fragments by limited reduction or from whole antibodies by digestion with papain in the presence of a reducing agent (generally, Paul, W., Ed., FUNDAMENTAL IMMUNOLOGY (See 2nd edition, Raven Press, NY, 1989, Chapter 7, all of which are incorporated by reference for all purposes). Fragments can also be produced by recombinant DNA techniques. A segment of nucleic acid encoding the selected fragment is produced by digestion of the full length coding sequence with restriction enzymes or by de novo synthesis. Fragments are often expressed in the form of phage coat fusion proteins.

Many of the above-described immunoglobulins are without loss of binding specificity or effector function, or loss of excessive binding affinity (ie, less than about 10 ⁶ M ⁻¹ or less than about 10 ⁷ M ⁻¹ ). Minor amino acid substitutions, additions or deletions can be received in both the variable and constant regions. Usually, immunoglobulins incorporating such modifications show substantial sequence identity to the reference immunoglobulin from which they are derived. Mutant immunoglobulins can be selected that have the same specificity and increased affinity compared to the reference immunoglobulin from which they are derived. Phage display technology provides a useful technique for selecting such immunoglobulins. See, for example, Dower et al., WO91 / 17271 McCatterty et al., WO 92/01047; and Huse, WO 92/06204.

  The antibody of the present invention can be used with or without modification. Often, the antibody is labeled by either covalently or non-covalently binding to a detectable label.

  The antibodies of the present invention can be purified using well-known methods. Total antibodies of the invention, their dimers, individual light and heavy chains, or other immunoglobulin forms are in accordance with standard procedures in the art including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Can be purified using the methods and reagents of the present invention (see generally, Scopes, PROTEIN PURIFICATION; PRINCIPLES ANDPRACTICE 3rd Edition, (Springer-Verlag, NY, 1993)). Preferred are substantially pure immunoglobulins of homogeneity of at least about 90-95%, or even 98-99%, or more.

  As a method for producing the polypeptide of the present invention, for example, a method of obtaining the polypeptide by culturing primary cultured cells or established cells that produce the polypeptide, and isolating or purifying it from the culture supernatant or the like. Can be mentioned. Alternatively, using a gene manipulation technique, a gene encoding the polypeptide is incorporated into an appropriate expression vector, and an expression host is transformed with the gene, and assembled from the culture supernatant or cell extract of the transformed cell. A replacement polypeptide can be obtained. The host cell is not particularly limited as long as it expresses a polypeptide having physiological activity, and various host cells conventionally used in genetic manipulation (for example, E. coli, yeast, animal cells, etc.) are used. It is possible. As long as the polypeptide derived from the cells thus obtained has substantially the same action as the native polypeptide, one or more amino acids in the amino acid sequence are substituted, added and / or deleted. The sugar chain may be substituted, added and / or deleted.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, a cationic region or a binding site of a substrate molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that still retains its original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and still can provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the terms “variant” and “variant” may be used interchangeably. A variant or variant refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, the orthologs of the present invention may also be useful in the present invention.

  “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. Addition of an amino acid means adding 1 or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” refers to a compound that is different from a peptide but is equivalent to the peptide in at least one chemical or biological function. Thus, peptide analogs include those in which one or more amino acid analogs are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (eg, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  As used herein, a nucleic acid form of a polypeptide refers to a nucleic acid molecule that can express the protein form of the polypeptide. As described above, a part of the sequence of the nucleic acid may be deleted or substituted by another base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and the number is increased as long as the intended function is retained in the variant having the substitution, addition or deletion. be able to. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

  As used herein, “agonist” refers to a factor that binds to the receptor of a certain biologically active substance (ligand) and exhibits the same (or similar) action as that substance has.

  As used herein, a “receptor” is a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands, wherein Thus, this complexation has a biological structure.

  As used herein, “oncostatin M receptor” is a polypeptide that binds reversibly and specifically to oncostatin M, and is encoded by the nucleic acid sequence of SEQ ID NO: 10, unless otherwise specified. Mouse protein, human protein encoded by the nucleic acid sequence of SEQ ID NO: 12, rat protein encoded by the nucleic acid sequence of SEQ ID NO: 14, and homologs and orthologs of the protein, and variants of these proteins, homologs and orthologs , And all allelic variants.

  The term “ligand” as used herein is a binding partner for a specific receptor. The ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological means.

  As used herein, “oncostatin M receptor” refers to a receptor that specifically binds to oncostatin M. A typical oncostatin M receptor is a heterodimer of the gp130 molecule and the oncostatin M receptor β chain (OSMRβ).

  In general, pain sensation is a sensation that occurs when a body part has a strong infringement such as injury or inflammation. However, in order to cause pain sensation, injury / inflammation is not essential, and the same sensation may occur even when there is no injury or inflammation. Therefore, as used herein, the term “pain” is a pain sensation caused by a strong nociception such as injury / inflammation, as well as a pain sensation caused by those strong nociceptions, but is a strong sensation such as injury / inflammation. Pain that occurs without infringement. Pain is broadly divided into acute pain, inflammatory pain, and neuropathic pain.

  As used herein, “neuropathic pain” is not associated with acute external stimuli (eg, thermal, chemical, or mechanical stimuli) and is also accompanied by in vivo inflammation. For example, pain caused by abnormalities or defects in nerve cells.

  As used herein, an “effective amount” for prevention or treatment refers to an amount that is recognized as being medically effective in prevention or treatment (or treatment). Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

  As used herein, “analgesic agent” refers to an agent for treating pain to reduce pain sensation. The pharmaceutical composition of the present invention acts as an analgesic.

  As used herein, “gene therapy” refers to therapy performed by introducing a foreign gene into a living body.

  As used herein, “gene transfer” refers to a natural, synthetic or recombinant desired gene or gene fragment in a target cell in vivo or in vitro, and the introduced gene has its function. Introducing to maintain. The gene or gene fragment introduced in the present invention includes a nucleic acid which is DNA, RNA or a synthetic analog thereof having a specific sequence. Also, as used herein, gene transfer, transfection, and transfection are used interchangeably.

  As used herein, “gene transfer vector” and “gene vector” are used interchangeably. “Gene transfer vector” and “gene vector” refer to a vector capable of transferring a target polynucleotide sequence into a target cell. “Gene transfer vector” and “gene vector” include, but are not limited to, vectors derived from viruses. Representative viral-derived vectors include, but are not limited to: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus.

  As used herein, “foreign gene” refers to a nucleic acid that is contained within a gene transfer vector and originates from a source other than the host cell into which the gene is transferred by the gene transfer vector. In one aspect of the invention, the foreign gene is a regulatory sequence suitable for expression of the gene introduced by the gene transfer vector (eg, a promoter, enhancer, terminator, and poly A addition signal required for transcription, and A ribosome binding site necessary for translation, start codon, stop codon, etc.). In another aspect of the invention, the foreign gene does not contain regulatory sequences for expression of the foreign gene. In a further aspect of the invention, the foreign gene is an oligonucleotide or a decoy nucleic acid.

  The foreign gene contained in the gene transfer vector is typically a DNA or RNA nucleic acid molecule, but the nucleic acid molecule to be introduced may include a nucleic acid analog molecule. The molecular species contained in the gene transfer vector may be a single gene molecular species or a plurality of different gene molecular species.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a regulatory gene that affects its expression. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.

  As used herein, a “fragment” of a nucleic acid molecule refers to a polynucleotide that is shorter than the total length of the reference nucleic acid molecule and has a length sufficient for the production of the pharmaceutical composition of the present invention. Therefore, a fragment in the present specification refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.

  As used herein, “operably linked” refers to transcriptional translational regulatory sequences (eg, promoters, enhancers, etc.) or translational regulatory sequences that have the expression (operation) of the desired sequence. To be placed under control. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, “biological activity” is used interchangeably with “functional property”. In this specification, “biological activity” and “functional property” refer to an activity that a certain factor (for example, nucleic acid) can have in vivo, and includes activities that exhibit various functions. Is done. For example, if a factor is a gene that encodes a growth factor, its functional properties include expressing the growth factor in a host cell, preferably promoting cell growth by its expression. For example, if a factor is a gene encoding an enzyme, its functional properties include allowing the enzyme activity to be expressed in a host cell, preferably making the enzyme activity detectable. In another example, when a factor is a gene encoding a ligand, the ligand expresses a ligand that binds to the corresponding receptor, and preferably the expression of the ligand causes cells having the receptor corresponding to the ligand to be expressed. Includes changing.

  As used herein, a “control region” of a gene refers to a region that controls the expression of the gene. Representative control regions include, but are not limited to, promoters, enhancers, terminators, and poly A addition signals, as well as ribosome binding sites required for translation, start codons, stop codons, and the like.

  As used herein, a “promoter” of a gene refers to a nucleic acid sequence capable of initiating transcription of a gene operably linked to that promoter. In the present invention, preferred promoters to be linked to cytotoxic genes are human oncostatin M receptor β chain gene promoter sequence (SEQ ID NO: 1), mouse oncostatin M receptor β chain gene promoter sequence (SEQ ID NO: 2), rat oncostat Statin M receptor β chain gene promoter sequence (SEQ ID NO: 3), or a fragment of these promoter sequences, which expresses an operably linked gene in a tissue specific manner similar to Oncostatin M receptor β chain. However, it is not limited to these.

  As used herein, “cytotoxic gene” refers to a gene that, when expressed in a host mammalian cell, indicates that host mammalian cell. As the cytotoxic gene, for example, the following various genes are well known: diphtheria toxin, ricin, and herpes thymidine kinase. Herpes thymidine kinase acts as a cytotoxic gene in the presence of cancyclovir.

  As used herein, a “kit” includes a plurality of containers and manufacturer's instructions, and each container includes a pharmaceutical composition of the present invention, other agents, and a carrier. A product.

  As used herein, a “subject” is a subject to which the pharmaceutical composition of the present invention is administered, including but not limited to animals such as humans, mice, cows, chickens, and the like. .

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or pharmaceutical adjuvants.

  As used herein, “cytotoxin” refers to an agent that, when applied to a cell, inhibits, suppresses, and / or delays cell proliferation and / or kills the cell. Various toxins are known and include, but are not limited to, saporin, diphtheria toxin, ricin, and herpes thymidine kinase.

  The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

(Preparation and use of retroviral vectors)
Retroviruses are used as vectors for foreign gene insertion. And the general parameters of this use are now quite well understood: retroviruses consist of a single-stranded RNA genome confined within the protein envelope. The genome itself, reading from the 5 ′ end to the 3 ′ end, includes: These are the cap, the 5 ′ translation region, the RNA section or packaging site designated “ψ” (this RNA section is required for RNA that can be packaged into proteins), and some proteins—its retro Coding sequence for viral nucleoprotein (gag): viral coat of reverse transcriptase and cranial protein (env) that promotes an intermediate step consisting of translation (pol) of DNA, and some 3 'untranslated All areas performed by the array. Three viral proteins are required for infection of the viral genome. This packaging site is necessary to produce the added infectious virus.

  Retroviruses undergo a “provirus” stage that includes a double-stranded cDNA copy of the RNA region encoding the protein. However, at this stage, the untranscribed 3 'and 5' regions are modified to contain a long terminal repeat (LTR) at either end of the cDNA encoding the protein. This long terminal repeat provides not only the appropriate promoter and enhancer sequences that result in transcription of the DNA, but also a sequence that terminates transcription at an operable position with respect to this coding portion.

  In normal infection, the proviral double-stranded cDNA can be absorbed into the genome of the host cell. Because of these effects, additional viral particle products containing the RNA genome were packaged in the protein capsule. In this procedure performed, the presence of the ψ package site in the provirus is critical.

The sequence encoding the retroviral protein could be replaced with the sequence for the desired protein to use the viral expression system when the modified virus infects host cells. . See, for example, U.S. Pat. No. 4,405,712 and Lang (supra). However, to achieve this, the modified viral genome requires a helper virus that can synthesize cranial proteins and package RNA transcription of foreign DNA.
Thus, for gene therapy, the proviral DNA form is inserted into an appropriate vector, copied, and packaged in a viral envelope with the help of a helper virus. For a general review, see Anderson, W. et al. F. Science (1984) 226: 401-409: Coffin, J. et al. , "Genome Structure", in RNA Tumor Viruses, Vol 2, Weiss et al, eds, 2d ed, (1985), Cold Spring Harbor, NY.

  The most commonly used retroviruses for gene therapy studies are murine sarcoma virus (MSV) or Moloney murine leukemia virus (MoMLV) (Mann. R. et al., Cell (1983) 33: 153-159). The proviral forms of these retroviruses are isolated and inserted into a nearly standard cellular cloning vector for amplification. Proviral insertions along the package site (including mRNA encoding gag, pol and env flanked by long terminal repeats containing control sequences) will result in a region containing RNA encoding this protein. Treated to replace the desired foreign gene. If this DNA is transfected into a host cell infected with a complete virus or a defective virus lacking only the packaging site, the RNA synthesized from the modified provirus will then be transferred to other cells. Packaged in virus particles for reinfection. This provides a mechanism of action for introducing the DNA encoding the desired active ingredient or drug into the cell by infection.

  There are two ways to get started. In one method, the modified proviral DNA is transfected into cells that resist infection from unmodified viruses that coexist in the cell. Normal viral vectors synthesize the packaging material and some of the mRNA produced by the modified provirus is packaged in a manner similar to normal virions. This can then be used to infect target cells for protein production. However, in addition to these collected virus envelopes, there will also be a certain number of repackaged normal viruses. If this virus is not isolated from a “feed-carrying” virus, it will easily cause further viral infections in host cells infected with the products of the virion production process.

  In a more useful method, a proviral cloning vector containing the desired gene is used to transfect genetically modified cells to produce a defective viral envelope (this defective viral envelope). Is actually an empty supply haulage). These cells are obtained by integration of a proviral form of a mutant retrovirus lacking the ψ package site. And several such cell lines are available for all techniques that seek them. These two series, designated ψ-1 or ψ-2, are Mann. R et al., Cell (1983) 33: 159-159, are described extensively. This is made by transfecting host NIH-3T3 fibroblasts with a plasmid containing the MoMLV proviral insert. From this insert, the ψ package site has been deleted. The ψ-2 cells clearly produce some empty viral envelope per cell that corresponds to the viral envelope of a unique virus within a generation. When these cells are transfected with proviral DNA containing both the foreign gene position and the package site (ψ), they are produced from proviral DNA containing the foreign gene so that they produce a modified virus. In these empty envelopes, a transcript of mRNA is packaged. This modified virus infects cells that are normally the host for MoMLV (in this case, Glycidae), however, this recombinant (modified virus) is present in the cell it “infects”. It should be noted that there is a defect in that it cannot cause the production of further modified virions (or other) virions. This recombinant can cause the production of the protein encoded by this gene in “infected” cells. However, the infection cannot spread additional cells because no additional virions are produced.

  More useful than ψ2 for the preparation of drugs for humans is the ψ-AM series. This series is described in Cone, R .; D. , Et al., Proc Natl Acad Sci USA (1984) 81: 6349-6353. These lines can also be obtained by transfecting NIH-3T3 cells. However, a vector named pMAV-ψ- must be used. This vector also contains a defective proviral insert lacking the ψ packaging site. However, pMAV-ψ- is a hybrid that encodes the MoMLV gag-pol sequence, and the coat sequence is derived from the amphoteric virus 4070A. The empty crusts produced by these cell lines package RNA transcripts of co-transfected and modified proviral DNA that produce pseudoviruses. This pseudovirus recognizes and infects human, field and mouse cells.

(Preparation and Use of Inactivated HVJ Envelope Vector Using Surfactant)
As an example of a viral vector, a method for preparing an HVJ envelope vector is known (Japanese Patent Laid-Open No. 2001-286282).

(1: Growth of HVJ)
HVJ can be generally used by inoculating a fertilized egg of a chicken by inoculation with a seed virus. However, cultured cells such as monkeys and humans, and a system for continuously infecting cultured tissues with a virus (cultivating a hydrolase such as trypsin). Any of those that are propagated by using a solution added to the liquid and those that are propagated by infecting a cultured cell with a cloned viral genome and causing persistent infection can be used.

  The growth of HVJ is performed as follows.

  HVJ seeds of HVJ grown using SPF (Specific pathogen free) fertilized eggs, separated and purified, are dispensed into cell storage tubes, and 10% DMSO is added and stored in liquid nitrogen And prepare.

  Chicken eggs immediately after fertilization are received and placed in an incubator (SHOWA-FURANKI P-03 type; accommodating about 300 chicken eggs) and reared for 10-14 days under conditions of 36.5 ° C. and humidity of 40% or more. In the dark room, use an ophthalmoscope (light bulb light exits through a window with an aperture of about 1.5 cm) to check embryo survival and air chamber and chorioallantoic membrane. Mark the location of virus injection with a pencil approximately 5 mm above (select the location excluding the thick blood vessels). Seed virus (taken from liquid nitrogen) 500 times with polypeptone solution (mixed with 1% polypeptone, 0.2% NaCl, adjusted to pH 7.2 with 1M NaOH, autoclaved and stored at 4 ° C) Diluted to 4 ° C. Eggs were sterilized with isodine and alcohol, and a small hole was made in a thousand holes at the site of virus injection. Inject 1 ml into the chorioallantoic cavity using a 1 ml syringe with a 26 gauge needle. Place melted paraffin (melting point 50-52 ° C.) on the hole using a Pasteur pipette to close it. Eggs were placed in an incubator and raised for 3 days under conditions of 36.5 ° C. and humidity of 40% or more. The inoculated eggs are then placed overnight at 4 ° C. The next day, the air chamber portion of the egg is split with tweezers, a 10 ml syringe with an 18 gauge needle is placed in the chorioallantoic membrane, the chorioallantoic fluid is aspirated, collected in a sterile bottle, and stored at 4 ° C.

(2: Purification of HVJ)
HVJ can be purified by purification methods by centrifugation, column purification methods, or other purification methods known in the art.
(2.1: Purification method by centrifugation)
Briefly, the grown virus solution was collected, and the tissue and cell debris in the culture solution and chorioallantoic fluid were removed by low speed centrifugation. The supernatant is purified by high-speed centrifugation (27,500 × g, 30 minutes) and ultracentrifugation (62,800 × g, 90 minutes) using a sucrose density gradient (30-60% w / v). It should be noted that the virus should be handled as gently as possible during purification and stored at 4 ° C.

  HVJ is purified by the following method.

  About 100 ml of HVJ-containing chorioallantoic fluid (collecting HVJ-containing chicken egg chorioallantoic fluid and storing at 4 ° C.) was placed in two 50 ml centrifuge tubes with a wide Komagome pipette (Saeki, Y., and Kaneda, Y: Protein modified liposomes (HVJ-1 iposomes) for the delivery of genes, volume of oligonucleotides and proteins, 27th edition, Cell biology; A. laboratory handbook, E. c. ~ 135, 1998), centrifuge at 3000 rpm for 10 minutes at 4 ° C (brake off) in a low speed centrifuge to remove egg tissue debris.

  After centrifugation, the supernatant is dispensed into four 35 ml centrifuge tubes (for high-speed centrifugation), and centrifuged at 27,000 g for 30 minutes using an angle rotor (accelerator and brake are on). The supernatant was removed, and about 5 ml of BSS (10 mM Tris-HCl (pH 7.5), 137 mM NaC1, 5.4 mM KC1; autoclaved and stored at 4 ° C.) (PBS instead of BSS) was added to the precipitate, It left still at 4 degreeC as it was. Gently pipette with a wide-bottom Komagome pipette to loosen the precipitate and collect in a single tube. Similarly, centrifuge at 27,000 g for 30 minutes with an angle rotor. Excluding the supernatant, about 10 ml of BSS was added to the precipitate, and the mixture was allowed to stand at 4 ° C. overnight. Gently pipette with a wide-bottom Komagome pipette to loosen the precipitate, and centrifuge at 3000 rpm for 10 minutes at 4 ° C (brake off) in a low-speed centrifuge (brake off). Exclude the tissue fragments and virus clumps that could not be removed. The supernatant is placed in a new sterile tube and stored as purified virus at 4 ° C.

  This virus solution 0. 0.9 ml of BSS is added to 1 ml, the absorption at 540 nm is measured with a spectrophotometer, and the virus titer is converted to hemagglutination activity (HAU). An absorption value 1 at 540 nm corresponded to approximately 15,000 HAU. HAU is considered to be approximately proportional to the fusion activity. In addition, erythrocyte agglutinating activity may be actually measured using chicken erythrocyte fluid (0.5%) (Animal Cell Utilization Manual, REALIZE INC. (Uchida, Oishi, Furuzawa) P259-268, 1984. See

Furthermore, purification of HVJ using a sucrose density gradient can be performed as necessary. Specifically, the virus suspension is placed in a centrifuge tube overlaid with 60% and 30% sucrose solutions (autoclaved), and density gradient centrifugation is performed at 62,800 × g for 120 minutes. After centrifugation, the band seen on the 60% sucrose solution layer is collected. The collected virus suspension is dialyzed overnight at 4 ° C. using BSS or PBS as an external solution to remove sucrose. If not used immediately, add glycerol (autoclave sterilization) and 0.5M EDTA solution (autoclave sterilization) to the virus suspension to a final concentration of 10% and 2-10 mM, respectively, and gently at −80 ° C. Frozen and finally stored in liquid nitrogen (cryopreservation is possible with 10 mM DMSO instead of glycerol and 0.5 M EDTA solution).
(2.2: Purification method by column and ultrafiltration)
Instead of the purification method by centrifugation, purification of HVJ using a column is also applicable to the present invention.

  Briefly, purification is performed using concentration by ultrafiltration with a filter having a molecular weight cut-off of 50,000 (about 10 times) and elution by ion exchange chromatography (0.3 M to 1 M NaCl).

  Specifically, in this example, HVJ is purified by a column using the following method.

  After the chorioallantoic fluid was collected, it was filtered through an 80 μm to 10 μm membrane filter. Add 0.006-0.008% β-propiolactone (BPL) (final concentration) to chorioallantoic fluid (4 ° C., 1 hour) to inactivate HVJ. BPL is inactivated by incubating chorioallantoic fluid at 37 ° C. for 2 hours.

Concentrate approximately 10 times by tangential flow ultrafiltration using 500 KMWCO (A / G Technology, Needham, Massachusetts). As a buffer solution, 50 mM NaCl, 1 mM MgCl ₂ , 2% mannitol, 20 mM Tris (pH 7.5) is used. The HAU assay gives excellent results with almost 100% HVJ recovery.

  HVJ is purified by column chromatography (buffer solution: 20 mM TrisHCl (pH 7.5), 0.2-1 M NaCl) by QSepharoseFF (Amersham Pharmacia Biotech KK, Tokyo). Generally, the recovery is 40-50% and the purity is 99% or more.

  The HVJ fraction is concentrated by tangential flow ultrafiltration using 500 KMWCO (A / G Technology).

(3: Inactivation of HVJ)
When inactivation of HVJ is necessary, as described below, UV irradiation or alkylating agent treatment is performed.

(3.1: UV irradiation method)
1 ml of the HVJ suspension was placed in a petri dish having a diameter of 30 mm and irradiated with 99 or 198 millijoules / cm ² . Gamma irradiation can also be used (5-20 gray), but complete inactivation does not occur.

(3.2: Treatment with alkylating agent)
Immediately before use, 0.01% β-propiolactone was prepared in 10 mM KH ₂ PO. During work, keep the temperature low and work quickly.

  Β-propiolactone was added to a suspension of HVJ immediately after purification to a final concentration of 0.01% and incubated on ice for 60 minutes. Incubate for 2 hours at 37 ° C. Dispense 10,000 HAU per tube into an Eppendorf tube, centrifuge at 15,000 rpm for 15 minutes, and store the precipitate at -20 ° C. Regardless of the inactivation method described above, the precipitate can be stored at -20 ° C., and the DNA can be directly incorporated by treatment with a surfactant to prepare a vector.

(4: Creation of HVJ envelope vector)
Add 92 μl of a solution containing 200 to 800 μg of foreign DNA to the preserved HVJ and well suspend by pipetting. This solution can be stored at −20 ° C. for at least 3 months. When protamine sulfate is added to DNA before mixing with HVJ, the expression efficiency is enhanced by a factor of 2 or more.

  This mixed solution was placed on ice for 1 minute, 8 μl of octyl glucoside (10%) was added, the tube was shaken on ice for 15 seconds, and left on ice for 45 seconds. The treatment time with the surfactant is preferably 1 to 5 minutes. In addition to octylglucoside, Triton-X100 (t-octylphenoxypolyethoxyethanol), CHAPS (3-[(3-colamidopropyl) -dimethylammonio] -1-propanesulfonic acid), NP-40 (nonylphenoxypoly) Surfactants such as ethoxyethanol may also be used. Preferred final concentrations of Triton-X100, NP-40 and CHAPS are 0.24-0.80%, 0.04-0.12% and 1.2-2.0%, respectively.

  1 ml of cold BSS was added and immediately centrifuged at 15,000 rpm for 15 minutes. Add 300 μl of PBS or physiological saline to the resulting precipitate and suspend by vortexing or pipetting. The suspension can be used directly for gene transfer or can be used for gene transfer after storage at −20 ° C. This HVJ envelope vector can maintain comparable gene transfer efficiency after storage for at least 2 months.

(Gene therapy)
For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExplorMorL. , Stockton Press, NY (1990).

  Nucleic acid constructs used for gene therapy can be administered either locally or systemically using well-known gene transfer vectors. Where such a nucleic acid construct includes a coding sequence for a protein, expression of the protein can be induced by use of an endogenous mammalian promoter or a heterologous promoter. The expression of the coding sequence can be constitutive or regulated.

  When various well-known gene transfer vectors are used as a composition for gene therapy, administration of the vector is performed by locally administering a vector suspension suspended in PBS (phosphate buffered saline) or saline. This can be done by direct injection (eg, in cancer tissue, liver, muscle, brain, etc.) or by administration into blood vessels (eg, intraarterial, intravenous, and portal vein).

  In one embodiment, the gene transfer vector is generally used in a unit dose injectable form (solution, suspension or emulsion) of the gene transfer vector (ie, a pharmaceutically acceptable carrier (ie, used). Non-toxic to recipients at different dosages and concentrations, and compatible with the other ingredients of the formulation). For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to gene transfer vectors.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphoric acid, citric acid, succinic acid, acetic acid, and other organic acids or their salts An antioxidant such as ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide (eg, polyarginine or tripeptide); a protein (eg, serum albumin, gelatin, or an immunoglobulin); a hydrophilic polymer ( Amino acids (eg, glycine, glutamic acid, aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (including cellulose or derivatives thereof, glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg mannitol) Other sorbitol); counterions (such as sodium); and / or nonionic surfactants (e.g., may include polysorbates, poloxamers), or PEG.

  A pharmaceutical composition comprising a gene transfer vector can typically be stored as an aqueous solution in unit or multi-dose containers, such as sealed ampoules or vials.

  A pharmaceutical composition comprising a gene transfer vector is a clinical condition of an individual patient (eg, a condition to be prevented or treated), in a manner consistent with good medical practice, of the composition comprising the gene transfer vector. It is formulated and administered taking into account the delivery site, target tissue, method of administration, dosing schedule and other factors known to those of skill in the art.

  For example, when an HVJ (Sendai virus) gene vector is administered to a mouse, a gene corresponding to 20 to 20,000 HAU, preferably 60 to 6,000 HAU, more preferably 200 to 2,000 HAU per mouse. A vector is administered. The amount of the foreign gene contained in the administered gene vector is 0.1-100 μg, preferably 0.3-30 μg, more preferably 1-10 μg per mouse.

  In addition, when an HVJ (Sendai virus) gene vector is administered to humans, it corresponds to 400-400,000 HAU, preferably 1,200-120,000 HAU, more preferably 4,000-40, per subject. A gene vector equivalent to 000 HAU is administered. The amount of the foreign gene contained in the administered gene vector is 2 to 2,000 μg, preferably 6 to 600 μg, more preferably 20 to 200 μg per subject.

(General method)
Molecular biological techniques, biochemical techniques, microbiological techniques, and glycoscience techniques used in this specification are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J. et al. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL
Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approc, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press; Method in
Enzymology 230, 242, 247, Academic Press, 1994; a separate volume of experimental medicine, “Gene Transfer & Expression Analysis Experimental Method”, Yodosha, 1997, etc., which are related in this specification (may be all) ) Is incorporated by reference.

(Prescription)
The invention also provides a method of treating and / or preventing a disease or disorder (eg, an infection) by administering an effective amount of a therapeutic agent to a subject. By therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).

  The therapeutic agent is considered to be a medical practice standard (GMP = good) taking into account the clinical condition of the individual patient (especially the side effects of the therapeutic agent alone treatment), the site of delivery, the method of administration, the dosage regimen and other factors known to those skilled in the art. Formulate and dose in a manner consistent with medical practice. Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

  As a general suggestion, the total pharmaceutically effective amount of the therapeutic agent administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient weight, as described above. This is left to therapeutic discretion. More preferably, for the cell bioactive substances of the present invention, this dose is at least 0.01 mg / kg / day, most preferably about 0.01 mg / kg / day and about 1 mg / kg / day for humans. Between. For continuous administration, typically the therapeutic agent is injected 1 to 4 times daily at a dosage rate of about 1 μg / kg / hour to about 50 μg / kg / hour or continuous subcutaneous infusion (eg, using a minipump). It is administered by either of the following. Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.

  The therapeutic agent can be oral, rectal, parenteral, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, instillation, or transdermal patch), oral or oral or It can be administered as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are oral, rectal, parenteral, intracisternally, intravaginally, intraperitoneally, topically (powder, ointment, gel, infusion, or transdermal patch) Etc.), or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, in acceptable quality oils). Or an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).

  Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl Acetate (Langer et al., Ibid.) Or poly-D-(-)-3-hydroxybutyric acid (EP133,988).

  Sustained release therapeutic agents also include the therapeutic agents of the present invention entrapped in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Disease of Infectious Disease and Cancer, Loop, -See Berstein and Fiddler (eds.), Liss, New York, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Pat. No. 4,485,045 And 4,544,545 and EP 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol, and the selected proportion is adjusted for optimal therapeutic agents.

  In still further embodiments, the therapeutic agents of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgary 88: 507 ( 1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)).

  Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  For parenteral administration, in one embodiment, in general, the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidation and other compounds known to be harmful to therapeutic agents.

  Generally, formulations are prepared by contacting the therapeutic agent uniformly and intimately with liquid carriers or finely divided solid carriers or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts. Buffering agents; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

  The therapeutic agent is typically formulated in such a vehicle at a pH of about 3-8 at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml. It is understood that by using the specific excipients, carriers or stabilizers described above, salts are formed.

  Any drug to be used for therapeutic administration may be in a state free of organisms / viruses other than viruses as an active ingredient, that is, in a sterile state. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). In general, the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper puncturable with a hypodermic needle.

  The therapeutic agents are typically stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous therapeutic agent and the resulting mixture is lyophilized. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration. Represents institutional approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.

  The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. The combinations can be administered, for example, either simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual . Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.

  In certain embodiments, the pharmaceutical compositions of the invention are administered in combination with non-steroidal anti-inflammatory analgesics and / or narcotics.

  In further embodiments, the therapeutic agents of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the therapeutic agents of the present invention include glucocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones , Salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzidoamine, bucolome, difenpyramide, ditazole, emolphazone, Guaiazulene, nabumetone, nimesulide, orgothein, oxaceptol, paraniline, perizoxal, pifoxime, prochiazone, proxazole, and tenidap That, but it is not limited to these.

  In further embodiments, the therapeutic agents of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

Example 1: Neuron development and pain behavior in Oncostatin M ^{− / −} mice
(Tissue preparation)
C57BL / 6J mouse embryos (11.5, 14.5, and 17.5 day embryos), female newborn mice (0, 7, and 14 days old), and female Adult mice (8 weeks old) were used (Nihon SLC, Hamamatsu City). Embryos were removed by caesarean section and immersed in ice-cold 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde (PFA) and left overnight at 4 ° C. Newborn and adult mice were perfused from the heart with ice cold 0.85% NaCl. After that, when immunohistochemical staining is performed, perfusion with ice-cold Zamboni fixative (2% PFA, 0.2% picric acid in 0.1M PB, pH 7.4) for in situ hybridization Were perfused with 4% PFA in 0.1 M phosphate buffered saline (PBS). All fixation by perfusion was performed using mice that had been deeply anesthetized with diethyl ether. DRG (L4 / L5) and spinal cord were rapidly removed and then postfixed at 4 ° C. for 3 hours and finally perfused with 20% sucrose in 0.1 M PBS. O. C. T.A. Embedded in medium (Miles, Elkhart, IN, USA) and quickly frozen in n-hexane on dry ice and stored at -80 ° C.

  All animal experiments were conducted under the control of the Animal Experiment Committee of Wakayama Medical University in accordance with the animal experiment guidelines of Wakayama Medical University and the notification of animal care of the Japanese government. All experiments were performed to minimize the number of animals used and the suffering suffered by the animals.

(Preparation of RNA probe)
A 275 bp Asp718-XhoI cDNA fragment (coding region) of the mouse oncostatin M receptor β chain gene was ligated into the pBluescriptII SK + (−) vector. To prepare the antisense probe, the ligated vector was cut with Asp718. The ligated vector was cut with XhoI to prepare a sense probe. For the preparation of the antisense probe, in vitro transcription was performed using T3 RNA polymerase. T7 RNA polymerase was used for the preparation of the sense probe, and in vitro transcription was performed. [Α- ³⁵ S] UTP was used for radiolabeling during probe preparation.

(In situ hybridization analysis)
In situ hybridization was performed as described in Tamura et al., Mech Dev 115: 127-131 (2002); Tamura et al., Neuroscience 119 (2003) 991-997. The experiment is briefly described below.

The frozen section was cut to a thickness of 6 μm using a cryostat. After hybridization for 16 hours at 55 ° C. with sense and antisense ³⁵ S-labeled cRNA probes, slides were immersed in Kodak NTB-2 liquid emulsion (Kodak, Rochester, NY, USA). The soaked autoradiogram was developed and fixed after 14 days. Sections were counterstained with hematoxylin and eosin and observed using bright and dark field microscopes (XF-WFL, Nikon, Tokyo).

(Immunohistochemical method)
Immunohistochemical staining was performed as described by Tamura et al., Mech Dev 115: 127-131 (2002); Tamura et al., Neuroscience 119 (2003) 991-997. 6 μm cryostat tissue sections from DRG (L4 / L5) and 20 μm sections of mouse spinal cord mounted on slides were processed by immunohistochemical techniques. Sections were preincubated for 1 hour at room temperature (RT) in 0.1 M PBS containing 10% healthy donkey serum, then primary antibody solution (0.5%
Incubation in fetal bovine serum albumin (BSA) and a solution containing 0.3% Triton X-100) at 4 ° C. overnight. Primary antibody was used at the following dilutions; goat anti-oncostatin M receptor β chain antibody 1:50 (Santa Cruz Biotechnology, Santa Cruz, California, USA); rabbit anti-VR1 antibody 1: 500 (Oncogene, San Diego, USA) California); rabbit anti-CGRP antibody 1: 1000 (Amersham, Arlington Heights, Illinois, USA); rabbit anti-TrkA antibody 1: 100 (Upstate Biotechnology, Lake Placid, NY, USA); rabbit anti-c-Ret antibody 1: 100 (Tamura et al., Tamura et al., Eur. J. Neurosci 17: 2287-2298 (2003)); guinea pig anti-P2X3 antibody 1: 1000 ( Neuromics, Minneapolis, New York, USA). The next day, sections were washed with 0.1 M PBS containing 0.1% Triton X-100 and a Cy2- / Cy3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) (2% Normal mouse serum and 1: 400 diluted antibody containing 0.3% Triton X-100) for 1 hour at room temperature. For the avidin-biotin complex method, after incubation with primary serum, the sections were treated with biotinylated donkey anti-goat IgG antibody (Jackson).
ImmunoResearch; used 1: 500 diluted in 0.5% BSA and 0.3% Triton X-100 solution) for 1 hour at room temperature. After washing with 0.1M PBS, sections were incubated with 7-amino-4-methylcoumarin-3-acetic acid conjugated streptavidin (Jackson ImmunoResearch; in 0.1M PBS) for 1 hour at room temperature. Immunofluorescence images were obtained with a Nikon VFM microscope using fluorescence attachment.

(Quantitative assay)
For stereological cell counting, L4 / L5 DRGs from 4 perfused mice were pooled and processed for sectioning. The entire DRG is made into 6 μm serial sections and NeuroTrace (Molecular Probe, Eugene, Oreg., USA) diluted 1: 500 for 20 minutes or 4 ′, 6-diamidino-2-phenylindole dihydrochloride Were stained for nuclear staining after incubation with the secondary antibody for 5 minutes. After staining, color digitized images of each section were obtained using an epifluorescent microscope (Eclipse E800, Nikon) equipped with a color 3 CCD camera (C5810, Hamamatsu Photonics, Hamamatsu City). The number of fluorescently labeled cells was counted under observer-blind conditions using Adobe Photoshop software (Adobe Systems, San Jose, Calif.).

  To obtain unbiased estimates using NeuroTrace for the entire spinal ganglion (DRG) neuron and for the entire VR1 or P2X3 labeled neuron, the neurons in the section are Counts (Coggeshall, (1992) Neurosci. 15: 9-13). Briefly, pairs of adjacent sections were compared in each DRG (reference section and index section). Within the measured area of this section, only those neurons with clearly visible nuclei (single and multiple nuclei) are counted in the reference section, and then the density of the neurons [number of cells / (measured Area × section thickness)]. For each DRG, every tenth section was analyzed, starting with randomly selected sections between 1 and 10.

The cross-sectional area covered by neurons was measured for DRG (NIH image, version 1.61), the area measured for the volume of each ganglion occupied by neurons (μm ³ × 10 ⁶ ), section thickness, and each Estimated from the total number of sections containing ganglia. Next, the total number of NeuroTrace-stained neurons and the number of VR1-labeled or P2X3-labeled neurons in the DRG was determined by multiplying the total DRG volume by the neuron density (Ganderson et al., Acta. Pathol. Microbiol. Immunol.Scand. 96: 379-394).

  To determine the size of the cells, the cells were manually outlined using a computer mouse, and the cross-sectional area of the outlined cell profile was quantified using NIH images (1.61). Next, a size-frequency histogram was generated from the counted neurons. Values shown are mean ± standard error. All statistical analyzes were performed using Student's t-test. Significant when p <0.05.

(Gene targeting of the oncostatin M locus)
Genomic clones that hybridize to mouse oncostatin M cDNA were obtained from a λ phage 129 / Sv mouse genomic library and the clones were partially digested with BamHI, MboI, BglII, or Sau3AI. A targeting vector is constructed by cloning a selected 15.8 kb clone (including the entire mouse oncostatin M locus) into the pJMM4 plasmid (including a neomycin resistance cassette driven by the phosphoglycerate kinase (PGK) promoter). did. The gene targeting vector is replaced by replacing the endogenous exon II and exon III coding regions and the intervening intron with the PGKNeo gene, and introducing diagnostic XbaI and SphI sites into the genomic locus. Designed.

  Progeny genotyping was performed using Southern analysis and / or PCR. For Southern analysis, genomic DNA was digested with SphI and hybridized with a 5 'external probe (600 bp long). The wild type allele was detected as a 9 kb band. On the other hand, the mutant allele produced a 7 kb band. Two sets of primers were used in the same reaction mixture for genotyping by PCR. Primer OSMs (CAA GGG AAC CCG CAG AAT CTG: SEQ ID NO: 16) and primer OSMa (TGA ATC AGC TTG TGT CAT CAG: SEQ ID NO: 17) are used to amplify a 420 bp fragment of the wild type Oncostatin M locus did. In order to amplify a 585 bp fragment from the Neo gene within the targeted locus, primers NEOs (GAA CAA GAT GGA TTG CAC GCA G: SEQ ID NO: 18) and primer NEOa (CCA TGA TAT TCG GCA AGC AG: SEQ ID NO: 19) was used. PCR conditions were 95 ° C. for 2 minutes, and 94 ° C. for 30 seconds, 59 ° C. for 1 minute, and 72 ° C. for 1 minute for 30 cycles, and 72 ° C. for 10 minutes extension.

OSM ^+/− male mice were backcrossed to C57BL / 6 female mice for at least 6 generations, and oncostatin M-deficient mice were mostly in the background of the same gene line. OSM ^{+ / +} and OSM ^{− / −} littermate mice generated by heterozygous crosses were used for all experiments.

(Behavioral test)
These experiments were conducted with the approval of the Animal Experiment Committee of Wakayama Medical University.

Chemical pain was assessed by capsaicin, α, β-methylene ATP (αβ-meATP), and the formalin test (Dickenson and Sullivan, (1987) Neurosci. Lett. 83: 207-211; Cao et al., (1998) Nature. 392: 390-394); Caterina et al. (2000) Science 288: 306-313; Tsuda et al. (2002) Eur. J. et al. Neurosci. 15: 1444-1450). Mice were acclimated to the test platform for 2 hours daily for 4 days prior to the test. On the day of testing, mice are placed on the test platform for 1 hour, after which in each test 10 μl capsaicin (Sigma-Aldrich, 1-5 mg in 10 ml saline / 10% ethanol / 0.5 Tween 80) 10 μl of αβ-meATP (Sigma-Aldrich, 10 μmol / l) and 10 μl of 5% formalin were administered to the plantar surface of the left foot. The time of either licking or lifting the injected paw was recorded. In the hot plate assay (Cao et al. (1998) Nature 392: 390-394; Caterina et al. (2000) Science 288: 306-313), mice were habituated to a hot plate apparatus initially set at 45 ° C. for 2 minutes. Let The mice were then placed on a hot plate (48 ° C. to 58 ° C.) and the response latency until the hind paw was licked or jumped was recorded. The cut-off time for the 52 ° C. hot plate was 60 seconds, and the cut-off time for the 58 ° C. hot plate was 30 seconds. Mechanical sensitivity was evaluated by the tail pinch test (Cao et al. (1998) Nature 392: 390-394). We measured the latency of response to intense mechanical stimuli with a clip applied to the tail. Response to visceral irritation was determined by abdominal injection following intraperitoneal injection of 0.6% acetic acid (5 ml / kg) (built-in pain with inflammation) or MgSO ₄ (120 mg / kg) (visceral visceral pain without inflammation) Rated as the number of extensions. The frequency of extension response was counted for 20 minutes for acetic acid and 5 minutes for MgSO ₄ . Motor function was evaluated using a rotarod treadmill (Ugo Basile, Comerio, Italy). Initially, mice were trained to walk on a rotating rod for 3 minutes at a speed of 4 rpm. For testing, the speed was set to 4 rpm and then accelerated to 40 rpm. The time until the mouse fell after the start of acceleration was recorded.

(result)
(Expression pattern of oncostatin M receptor β chain in the development of DRG neurons)
To test the expression pattern of the oncostatin M receptor β chain in the development of DRG neurons, hybridization was performed using a small number of DRGs at the developmental stage (day 14.5 embryo to day 14 post birth) The expression of statin M receptor β chain mRNA in neurons was identified. Oncostatin M receptor β chain mRNA was not expressed in any cells by DRG at the embryonic stage (data not shown). Oncostatin M receptor β chain mRNA is detected in a small number of cells for the first time on the 10th day after birth, increases gradually, and reaches the expression level in adults on the 14th day after birth (FIG. 1).

(Decrease in pain DRG neurons in adult oncostatin M ^{− / −} mice)
To find the role of oncostatin M in the development of mouse DRG, we used oncostatin M-deficient mice. Oncostatin M ^{− / −} mice (also referred to as OSM ^{− / −} mice) develop normally and at any time during their development, with respect to the size or weight of the mouse, + / + littermates Or +/- litters could not be distinguished (data not shown). In adult mice, approximately 13% of total DRG neurons are oncostatin M receptor β chain positive small neurons that expressed VR1 and P2X3. Immunohistochemical staining for sensory neuron markers is VR1 positive (OSM ^{+ / +} , 46.8 ± 1.4%; OSM ^{− / −} , 24.0 ± 1.0%; p <0.001) DRG neurons , And P2X3 positive (OSM ^{+ / +} , 56.3 ± 0.9%; OSM ^{− / −} , 46.5 ± 1.2%; p <0.001) DRG neurons are adult oncostatin M ^{− / -} in the DRG, it showed that significantly decreased (Figure 2). No significant changes were observed for TrkA positive neurons and c-Ret positive neurons. No changes in CGRP positive DRG neurons were also observed in adult oncostatin M ^{− / −} mice (FIG. 2). Furthermore, size-frequency analysis of total neurons, VR1 positive neurons, and P2X3 positive neurons showed a decrease in small size neurons expressing VR1 and P2X3 in the DRG of Oncostatin M ^{− / −} mice (FIG. 3).

All oncostatin M receptor β chain positive neurons were VR1 / P2X3 positive. VR1 and P2X3 double immunofluorescence staining revealed a significant reduction of VR1 / P2X3 double positive neurons in DRG in Oncostatin M ^{− / −} mice (FIG. 4, arrows; OSM ^{+ / +} , 26. 4 ± 1.8%; OSM ^{− / −} , 7.8 ± 1.3%; p <0.01). Furthermore, oncostatin M receptor β chain positive DRG neurons were also reduced in oncostatin M ^{− / −} mice (OSM ^{+ / +} , 15.2 ± 1.8%; OSM ^{− / −} , 7.6 ± 0.6%; p <0.03). This indicates a reduction in VR1 / P2X3 / Oncostatin M receptor β chain triple positive neurons.

To determine in which layer VR1 and P2X3 are localized in the lumbar spinal cord of adult Oncostatin M ^{− / −} mice, we performed double-immunofluorescent staining for VR1 and P2X3. It was. Primary afferent fibers that express VR1 (FIG. 5, green) and P2X3 (FIG. 5, red) are known to be distributed in layers I-II and II (layer IIi), respectively. . As shown in FIG. 5, almost no VR1 / P2X3 double positive fibers could be detected in the IIi layer of the spinal cord of Oncostatin M ^{− / −} mice (yellow). This is in contrast to the detection of VR1 / P2X3 double positive fibers in the IIi layer of the spinal cord of Oncostatin M ^{+ / +} mice.

(Deficiency of pain sensitivity in adult Oncostatin M ^{− / −} mice)
Oncostatin M ^{− / −} mice did not show any defects in motor coordination. Retention times using the Rotorrod test were 98 ±. For Oncostatin M ^{+ / +} mice (also referred to as OSM ^{+ / +} mice). 1 second and 104 ± 4.7 seconds for Oncostatin M ^{− / −} mice (n = 6 per group).

To experimentally evaluate nociceptive function, we performed a behavioral battery test on adult oncostatin M ^{− / −} mice (FIG. 6). Initially, pain responses to chemical stimuli including capsaicin, αβ-meATP, and formalin were evaluated. This is because VR1 positive neurons and P2X3 positive neurons are reduced as shown in FIGS. As predicted from the above results in the present invention, when capsaicin stimulation was performed, Oncostatin M ^{+ / +} mice and Oncostatin M ^+/− mice (also referred to as OSM ^+/− mice) were vigorous. licked. However, in the case of Oncostatin M ^{− / −} mice, the behavioral response to capsaicin was significantly reduced (FIGS. 6A and B). Furthermore, pain behavior (licking) caused by injection into the hind paw of αβ-meATP (FIG. 6C) or formalin (FIG. 6D) was also significantly reduced in Oncostatin M ^{− / −} mice.

Next, the thermal response behavior in Oncostatin M ^{+ / +} mice and Oncostatin M ^+/− mice was compared. This is because VR1 is known to be a sensor for harmful heat (Cao et al. (1998) Nature 392: 390-394). The latency of response to sudden heat stimulation was measured using 52 ° C and 58 ° C hot plates. At both 52 ° C. and 58 ° C., Oncostatin M ^{− / −} mice showed significantly longer response latencies compared to Oncostatin M ^{+ / +} mice (FIG. 6E). Furthermore, the latency of response to more intense mechanical stimuli (tail clip) was also significantly longer in Oncostatin M ^{− / −} mice (FIG. 6F).

To investigate whether the decrease in pain response in Oncostatin M ^{− / −} mice occurs in the case of adverse stimuli to other tissues as well, intraperitoneal injection of MgSO ₄ (immediate type, independent of inflammation) Acute visceral pain behavior was evaluated by visceral pain response (resulting in abdominal agony) and intraperitoneal injection of acetic acid (inducing secondary pain to inflammatory response). Oncostatin M ^{− / −} mice had a marked reduction in the number of abdominal agony compared to wild type in both studies of visceral pain (FIGS. 6G and H).

  Thus, the acute nociceptive threshold in behavior was significantly affected by the absence of Oncostatin M.

(Discussion)
Although the LIF receptor β chain was expressed in various cells of the nervous system, no abnormal development of the nervous system has been reported in LIF ^{− / −} mice. LIF ^{− / −} mice showed female sterility due to abnormal implantation of undifferentiated germ cells, postnatal growth retardation, and defects in hematopoiesis and thymocyte proliferation (Escary et al. (2000) Nature 363: 361). -364). In Oncostatin M ^{− / −} mice, VR1 / P2X3-double positive neurons were significantly reduced. This result indicates that oncostatin M binding to the oncostatin M receptor is essential for the generation of normal amounts of VR1 / P2X3-double positive neurons. Therefore, VR1 / P2X3-double positive neurons can also be significantly reduced by administering an antagonist of Oncostatin M to the living body.

Although the exact mechanism by which Oncostatin M deficiency causes a marked decrease in VR1 / P2X3-double positive neurons is not clear, the following possibilities are considered: One is the possibility that Oncostatin M regulates the expression of VR1 and P2X3. VR1 and P2X3 expression in DRG has been reported to be increased by peripheral inflammation (Ji et al., (2002) Neuron 36: 57-68; Xu and Huang, (2002) Neurosci., 22: 93- 102). Furthermore, oncostatin M is produced by activated T cells, macrophages, polymorphonuclear neutrophils, and eosinophils that are involved in local inflammation (Modur et al. (1997) J. Clin. Invest. 100: 158-168; Grenier et al. (1999) Blood 93: 1413-1421; Wallace et al. (1999) J. Immunol. 162: 2247-5555; Tamura et al. (2002) Dev.Dyn.225: 327-331. ). These findings suggest that Oncostatin M regulates VR1 and P2X3 expression in nociceptive neurons. Another possibility is that Oncostatin M can act as a postnatal survival factor for certain parts of nociceptive neurons. Indeed, the ratio of small size neurons to total DRG neurons was significantly reduced in Oncostatin M ^{− / −} mice. Although the exact mechanism by which Oncostatin M deficiency causes a marked decrease in VR1 / P2X3-double positive neurons is unknown, the experimental results described herein show that Oncostatin M antagonists are It shows that VR1 / P2X3-double positive neurons can be significantly reduced by administration.

Consistent with histological results, the experimental results described herein show that Oncostatin M ^{− / −} mice have a significant adverse response in models of acute thermal pain, chemical pain, and visceral pain. Demonstrated that it was reduced. This result indicates that Oncostatin M is required for an adverse response to acute pain and that the adverse response to acute pain is significantly reduced when Oncostatin M stimulation to the Oncostatin M receptor is reduced. Thus, pain can be treated with agents containing antagonists of Oncostatin M.

Furthermore, the pain treatment effect is not only brought about by blocking oncostatin M receptor stimulation by oncostatin M using an antagonist of oncostatin M, but also selectively removing cells expressing oncostatin M receptor. Also brought by. This is because the selective removal of cells expressing Oncostatin M receptor is similar to Oncostatin M ^{− / − in} that it does not bring about physiological effects due to Oncostatin M stimulation.

Interestingly, a reduced response to mechanical pain was also observed in Oncostatin M ^{− / −} mice. In contrast, VR1 ^{− / −} mice or P2X3 ^{− / −} mice responded normally to mechanical pain (Caterina et al. (2000) Science 288: 306-313; Souslova et al. (2000) Nature 407: 1015). -1017). The exact mechanism to explain this result is unknown, but this result suggests that a decrease in stimulation by oncostatin M or a decrease in oncostatin M receptor-expressing cells can also reduce the response to mechanical stimulation as well. Indicates.

  The results presented herein above show that oncostatin M plays an important role in the postnatal phase of assembly of neural pathways that detect harmful stimuli. Furthermore, these results indicate that Oncostatin M antagonists can be used as analgesics targeting Oncostatin M receptor β chain positive neurons.

Example 2: Treatment of pain with an oncostatin M antagonist
The results of Example 1 showed that oncostatin M antagonist can be used to treat pain. To confirm this, mice are actually administered with oncostatin M antagonist and the changes in pain behavior in the mice are examined.

(Preparation of oncostatin M antagonist)
As the oncostatin M antagonist used in this Example, an anti-oncostatin M antibody or an anti-oncostatin M receptor antibody is selected. The amount of antibody to be used varies depending on the affinity of the antibody, but an antibody solution having a concentration of 1-1000 μg / ml is usually used. This antibody solution is dissolved in phosphate buffered saline and 5-20 μl is administered subcutaneously or intrathecally in mice.

(Reduced pain sensitivity by oncostatin M antagonist)
It is confirmed that mice receiving oncostatin M antagonists show no defects in motor coordination. This is confirmed by the holding time using the rotor rod test.

  To experimentally evaluate nociceptive function, we perform a behavioral battery test on mice treated with oncostatin M antagonist. Next, pain responses to chemical stimuli including capsaicin, [alpha] [beta] -meATP, and formalin are evaluated in both oncostatin M antagonist treated and non-treated mice. As predicted from the above results in the present invention, when capsaicin stimulation is performed, mice that do not receive Oncostatin M antagonist lick their feet vigorously, but mice that receive Oncostatin M antagonist do not lick their feet. Decreases. This indicates that behavioral responses to capsaicin are significantly reduced in mice treated with oncostatin M antagonist. Further, when αβ-meATP or formalin is used, the same results as in capsaicin are obtained.

  Next, the thermal response behavior in mice receiving and receiving no oncostatin antagonist is compared. Specifically, the latency of response to a sudden thermal stimulus is measured using hot plates at 52 ° C and 58 ° C. At both 52 ° C. and 58 ° C., mice that receive the oncostatin M antagonist show significantly longer response latencies compared to mice that do not. Furthermore, the latency of response to more intense mechanical stimuli (tail clips) is also significantly increased in mice receiving Oncostatin M antagonist.

  This also indicates that the threshold of acute nociception in behavior is significantly affected by the administration of oncostatin M antagonist.

(Example 3: Treatment of pain by selective removal of oncostatin M receptor-expressing cells)
From the results of Example 1, it was shown that pain can be treated by reducing the oncostatin M receptor-expressing cells. In order to confirm this, gene therapy is actually performed on mice, and oncostatin M receptor-expressing cells are selectively removed, and changes in pain behavior in mice are examined.

  In this example, in order to selectively remove Oncostatin M receptor-expressing cells, a diphtheria toxin gene, which is a cytotoxic gene, is linked downstream of the promoter of the human Oncostatin M receptor β chain gene, and replication deficiency is found. A recombinant retroviral vector is constructed by linking to a retroviral vector, and the cytotoxic gene is specifically expressed in cells expressing the oncostatin M receptor β chain, and the cells are killed.

(Construction of gene therapy vector for selectively removing cells expressing Oncostatin M receptor)
The human oncostatin M receptor β chain gene promoter sequence of SEQ ID NO: 1 is used as a promoter region for specifically expressing a cytotoxic gene in cells expressing the oncostatin M receptor β chain. A diphtheria toxin gene is used as a cytotoxic gene linked to this promoter sequence.

(Preparation of gene therapy vector by transfection of packaging cells)
Specific methods of viral vector preparation by transfection of packaging cells are, for example, “Experimental Medicine Separate Volume, Featured Bio-Experiment Series, Successful Gene Transfer and Expression Analysis Protocol, Kazunori Nakajima / Yoshihiro Kitamura, Yodosha And “Experimental Medicine Separate Volume, Revised 4th Edition, New Genetic Engineering Handbook, Masaaki Muramatsu, Masaru Yamamoto, Yodosha”. Specifically, it is as follows.

PLAT-E cells are used as packaging cells. The PLAT-E cells are gently washed with PBS, 2 ml of PBS (with 0.1% trypsin and 1 mM EDTA) is added, and left at 37 ° C. for 10 minutes. Gently peel off the cells and suspend in DMEM (+ 10% FCS) solution. Centrifuge at 1500 rpm for 5 minutes, carefully suspend the cell pellet and seed 2 × 10 ⁶ cells in a 60 mm culture dish. After 12 to 16 hours, add 3 μg of DNA and 9 μl of FuGene (Roche) in 100 μl of DMEM and mix well. After standing at room temperature for 5 minutes, the DNA solution is added dropwise to a culture dish in which PLA-E cells are cultured, and gently mixed. After culturing for 24 hours, the culture medium is gently changed. After further culturing for 24 hours, the supernatant is collected, centrifuged at 4 ° C. and 1500 rpm for 5 minutes, and the supernatant is centrifuged again at 4 ° C. and 3000 rpm for 5 minutes. Use a stock solution.

(Gene therapy vector administration)
The above gene therapy vector stock is administered 5-20 ml subcutaneously or intrathecally in mice. One to two weeks after administration, the same test as the behavioral test described in Example 1 is performed.

(Reduction of oncostatin M receptor β chain expression neurons by retroviral vectors)
Using the same procedure as in Example 1, the expression pattern of the oncostatin M receptor β chain is tested. Hybridization is performed using mouse brain to identify expression of oncostatin M receptor β chain mRNA in neurons. In mice introduced with a retroviral vector, mRNA of oncostatin M receptor β chain in the whole brain is decreased. This indicates that introduction of a retroviral vector selectively removes oncostatin M receptor-expressing cells and reduces the proportion of cells expressing oncostatin M receptor β chain.

(Reduced pain sensitivity by selective removal of oncostatin M receptor-expressing cells)
It is confirmed that mice receiving retroviral vectors do not show any defects in motor coordination. This is confirmed by the holding time using the rotor rod test.

  To experimentally evaluate nociceptive function, we perform a behavioral battery test on retroviral vector-treated mice. Next, pain responses to chemical stimuli including capsaicin, [alpha] [beta] -meATP, and formalin are evaluated in both retroviral vector treated and non-treated mice. As predicted from the above results in the present invention, when capsaicin stimulation was performed, mice not receiving Oncostatin M antagonist lick their feet vigorously, but mice receiving Oncostatin M antagonist administration lick their feet The frequency decreases. This indicates that behavioral responses to capsaicin are significantly reduced in mice treated with oncostatin M antagonist. Further, when αβ-meATP or formalin is used, the same results as in capsaicin are obtained.

  Next, the thermal response behavior in mice receiving and not receiving retroviral vectors is compared. Specifically, the latency of response to a sudden thermal stimulus is measured using hot plates at 52 ° C and 58 ° C. At both 52 ° C. and 58 ° C., mice that receive the retroviral vector show significantly longer response latencies compared to mice that do not. Furthermore, the latency of response to more intense mechanical stimuli (tail clips) is also significantly increased in mice receiving retroviral vectors.

  This also indicates that the acute nociceptive threshold in behavior is significantly affected by the administration of retroviral vectors.

(Example 4: Treatment of pain by selective removal of oncostatin M receptor-expressing cells)
From the results of Example 1, it was shown that pain can be alleviated and cured by reducing nerves related to pain. Therefore, next, in a neuropathic pain model mouse, a treatment effect by reducing nerves related to pain was confirmed.

  As a neuropathic pain model mouse, a neurological pain model (CCI model) that induces allodynia was created by ligating the fifth lumbar nerve of the mouse with silk thread. The expression of ATF3, which is said to be induced in dorsal root ganglion neurons when allodynia was induced, was examined by immunohistochemical staining (Fig. 7, upper panel). In normal C57BL / 6 mice, almost no ATF3 expression was observed in neurons in the non-operative dorsal root ganglion, but very many ATF3 expressions were observed in neurons in the dorsal root ganglion on the operation side. It was. Furthermore, in order to study allodynia behaviorally, a von Frey test was performed (FIG. 7, lower row), and the force (grams) applied when starting escape behavior was significantly reduced (left side: on the operation side). Results). From this, it was confirmed that allodynia was induced histologically and behaviorally by this model.

  Next, changes in the spinal cord in the CCI model were examined. When immunostaining was performed with an antibody against Iba1, which is a marker for microglia, an increase in microglia was observed in the dorsal horn of the spinal cord on the surgical side (Fig. 8, upper), and further immunostaining was performed with an antibody against Oncostatin M (OSM). However, an increase in staining was also observed in the posterior horn of the spinal cord on the operation side (FIG. 8, lower row). This confirmed an increase in the expression of Iba1 and Oncostatin M in the CCI model.

  Next, a CCI model was created in a deficient (OSMRKO) mouse lacking both alleles of the oncostatin M receptor, and changes in the spinal cord were examined. From immunostaining with an antibody against Iba1, a microglial marker, surgery was performed. Increased microglia were observed in the dorsal horn of the spinal cord (CCI model side), and from the immunostaining with an antibody against Oncostatin M (OSM), increased staining was also observed in the dorsal horn of the surgical side (FIG. 9). , Top). However, in order to study allodynia behaviorally, a von Frey test was conducted (Fig. 9, lower), and the escape when stimulating the sole on the surgical side and the sole on the non-surgical side was escaped. There was no difference in the force required to initiate behavior (grams) and the response latency to escape behavior, and allodynia was controlled behaviorally. From these facts, it is shown that allodynia which is a kind of neuropathic pain is suppressed by deficiency of oncostatin M receptor positive neurons. This indicates that neuropathic pain can be treated by reducing oncostatin M receptor positive neurons.

  Then, next, the monoclonal antibody with respect to the extracellular domain of oncostatin M receptor was created, and the effect by anti-oncostatin M receptor antibody was confirmed. The resulting 5 types of anti-oncostatin M receptor rat antibodies were made into cocktails (5 micrograms of protein), mixed with saporin-conjugated anti-rat immunoglobulin antibodies (3.6 micrograms) and administered subcutaneously to mice. did. As a result, a decrease in oncostatin M receptor-positive neurons was observed (FIG. 10).

  From these, administration of anti-oncostatin M receptor antibody results in a decrease in oncostatin M receptor positive neurons, and neuropathic pain (allodynia) is treated by a decrease in oncostatin M receptor positive neurons. Thus, it was demonstrated that pain can be treated with an anti-oncostatin M receptor antibody.

(Example 5: Treatment of pain by administration of Oncostatin M)
One possible mechanism for the development of allodynia is abnormal extension of dorsal root ganglion neurons in the spinal cord. This abnormal progression is thought to be the result of an abnormality in the production of neurite inducers that should be originally produced in the spinal cord. However, it has not been specifically demonstrated.

  When immunostaining was performed with an antibody against Oncostatin M (OSM) in the spinal cord of the CCI model, increased staining was observed in the dorsal horn of the spinal cord on the surgical side (Fig. 8, bottom). It was estimated that one of the neurite inducers to be oncostatin M was. If Oncostatin M is a neurite attractant, Oncostatin M allows control (eg, suppression) of neurite germination. As a method for this suppression, neutralizing antibody of oncostatin M is injected intrathecally, or high concentration of oncostatin M is injected into the peripheral side of the neurite of the dorsal root ganglion neurons, It is envisaged to increase the concentration of oncostatin M. The former is relatively common, but there is also a point that the patient is greatly invaded when considering clinical application. Therefore, 3 days after the creation of the CCI model, a high concentration of Oncostatin M (250 microgram / 10 microliter) was injected into the muscle around the sciatic nerve of the mouse. As a result, allodynia was suppressed 2 days after the injection (FIG. 11). This result could not have been predicted at all before the present invention.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The compositions of the present invention make it possible to treat pain, particularly treatment-resistant pain such as cancer pain, neuropathic pain, inflammatory pain and the like.

FIG. 1 shows the oncostatin M receptor β chain gene mRNA in the DRG at each stage of development (PN0 = day 0 after birth; PN7 = day 7 after birth; PN14 = day 14 after birth; and adult). The result of the in situ hybridization which detects is shown. FIG. 2 shows a series of sections on adult lumbar DRG for the expression of sensory neuron markers on DRG in Oncostatin M ^{− / −} mice, for Oncostatin M ^{+ / +} and Oncostatin M ^{− / −} mice. . The scale bar is 100 μm. FIG. 3 is a size-frequency histogram showing the cross-sectional area distribution of total DRG neurons (A), VR1-positive DRG neurons (B), and P2X3-positive DRG neurons (C). These neurons were prepared from 4 Oncostatin M ^{+ / +} and Oncostatin M ^{− / −} mice. Except for small size neurons (<300 μm ² ), no significant difference was observed between the two genotypes. *, P <0.05, n = 4. FIG. 4 shows the results of VR1 and P2X3 double immunofluorescence staining in adult DRG of Oncostatin M ^{+ / +} mice (left panel), Oncostatin M ^{− / −} mice (right panel). VR1 / P2X3-double positive cells (arrows) in the combined image are significantly reduced. The scale bar is 100 μm. FIG. 5 shows the results of VR1 and P2X3 double immunofluorescence staining in the backbones of Oncostatin M ^{+ / +} adult mice (left panel) and Oncostatin M ^{− / −} adult mice (right panel). In Oncostatin M ^{+ / +} mice, strong double immunoreactivity (yellow) was localized inside the II layer of the spine. However, almost no double immunostaining of the spine was observed in Oncostatin M ^{− / −} mice. The scale bar is 200 μm. FIG. 6 shows the results showing a decrease in sensitivity to nociceptive stimuli in Oncostatin M ^{− / −} mice. (AD) Chemical nociceptive testing. (A) Delay in licking in response to intraplantar injection of capsaicin (1.0 μg; n = 6) compared to wild type mice (white bars) and heterozygous mice (hatched bars). Were significantly reduced in mutant mice (black bars); the last two groups are not different. (B) Delay in licking in response to the dose of capsaicin shown in the figure (n = 5). (C) Delay in licking action in response to αβ-meATP (n = 6). (D) Cumulative time (n = 6) of licking hind legs after subcutaneous injection of formalin. Phase I, 0-10 minutes after injection; Phase II, 15-50 minutes after injection. (E) Latency of licking action or jump in hot plate assay. (F) Latency of response to acute mechanical stimulus by tail clip. (G, H) Visceral pain response (agony) produced by intraperitoneal injection (G) of MgSO ₄ (G) or intraperitoneal injection of acetic acid (H) (n = 8-9 for both wild type and mutant). NS = no significant results; *, p <0.05; **, p <0.02; ***, p <0.01; ***, p <0.001. The upper photograph in FIG. 7 is a photograph in which the expression of ATF3 was examined by immunohistochemical staining. The left side is the dorsal root ganglion neuron that has undergone surgery and the right side is the dorsal root ganglion neuron that has not undergone surgery. The lower graph of FIG. 7 shows the results of the von Frey test. The vertical axis shows the stimulus applied to the sole when the escape action is started, in grams. Lt indicates the result of the left foot which is the CCI model, and Rt indicates the result of the right foot which is the control. The upper photograph in FIG. 8 shows the result of immunostaining with anti-Iba1 antibody in the CCI model. The lower photograph in FIG. 8 shows the results of immunostaining with anti-oncostatin M antibody in the CCI model. The upper photograph in FIG. 9 shows the results of immunostaining with anti-Iba1 antibody in a CCI model prepared from an oncostatin M receptor gene knockout mouse. The middle photograph in FIG. 9 shows the result of immunostaining with anti-oncostatin M antibody in a CCI model prepared from an oncostatin M receptor gene knockout mouse. The lower graph of FIG. 9 shows the results of the von Frey test. The vertical axis shows the stimulus applied to the sole when the escape action is started, in grams. Lt indicates the result of the left foot which is the CCI model, and Rt indicates the result of the right foot which is the control. The upper photograph in FIG. 10 shows the results of staining oncostatin M receptor positive cells when anti-oncostatin M rat antibody and saporin-conjugated anti-rat immunoglobulin antibody were administered. The photograph at the bottom of FIG. 10 shows the staining results of oncostatin M receptor-positive cells when anti-oncostatin M rat antibody and saporin-conjugated anti-rat immunoglobulin antibody were not administered. Lt indicates the result of the left foot which is the CCI model, and Rt indicates the result of the right foot which is the control. The result of intramuscular injection of oncostatin M is shown for the CCI model. The graph is the result of the von Frey test. The vertical axis shows the stimulus applied to the sole when the escape action is started, in grams.

SEQ ID NO: 1 = human oncostatin M receptor β chain gene promoter SEQ ID NO: 2 = mouse oncostatin M receptor β chain gene promoter sequence SEQ ID NO: 3 = rat oncostatin M receptor β chain gene promoter sequence SEQ ID NO: 4 = mouse oncostatin M SEQ ID NO: 5 = amino acid sequence of mouse oncostatin M SEQ ID NO: 6 = cDNA sequence of human oncostatin M SEQ ID NO: 7 = amino acid sequence of human oncostatin M SEQ ID NO: 8 = cDNA sequence of rat oncostatin M SEQ ID NO: 9 = Amino acid sequence of rat oncostatin M SEQ ID NO: 10 = cDNA sequence of mouse oncostatin M receptor SEQ ID NO: 11 = amino acid sequence of mouse oncostatin M receptor SEQ ID NO: 12 = cDNA sequence of human oncostatin M receptor SEQ ID NO: 13 = human Oncostatin M receptor amino acid sequence SEQ ID NO: 14 = rat Oncostatin M receptor cDNA sequence SEQ ID NO: 15 = rat Oncostatin M receptor amino acid sequence SEQ ID NO: 16 = primer OSMs
SEQ ID NO: 17 = primer OSMa
SEQ ID NO: 18 = Primer NEOs
SEQ ID NO: 19 = Primer NEOa

Claims (71)

A pharmaceutical composition for treating pain comprising an oncostatin M antagonist and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant. The pharmaceutical composition of claim 2, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody. 4. The pharmaceutical composition according to claim 3, wherein the anti-oncostatin M receptor antibody forms a conjugate with a cytotoxin. The pharmaceutical composition according to claim 4, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase. The pharmaceutical composition according to claim 1, wherein the oncostatin M antagonist is contained so that the local concentration at the site of administration is 0.1-100 mg / ml. 2. The pharmaceutical composition of claim 1, further comprising an anti-oncostatin M antagonist antibody conjugated to a cytotoxin. The pharmaceutical composition according to claim 1, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. The pharmaceutical composition according to claim 1, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. The method further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. A pharmaceutical composition according to 1. Use of an oncostatin M antagonist in the manufacture of a medicament for treating pain. Use according to claim 11, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant. 13. Use according to claim 12, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody. 14. Use according to claim 13, wherein the anti-oncostatin M receptor antibody is conjugated to a cytotoxin. 15. Use according to claim 14, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase. The use according to claim 11, which is contained in the medicament so that the local concentration at the site where the oncostatin M antagonist is administered is 0.1-100 mg / ml. 12. Use according to claim 11, further comprising the use of an anti-oncostatin M antagonist antibody conjugated to a cytotoxin. 12. Use according to claim 11, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. A kit for treating pain comprising the pharmaceutical composition according to claim 1. 20. The kit of claim 19, wherein the oncostatin M antagonist is selected from the group consisting of:
Anti-oncostatin M antibody, anti-oncostatin M receptor antibody, oncostatin M receptor fragment, fusion protein comprising at least part of oncostatin M receptor, anti-gp130 antibody, and oncostatin M variant. 21. The kit of claim 20, wherein the oncostatin M antagonist is an anti-oncostatin M receptor antibody. 24. The kit of claim 21, wherein the anti-oncostatin M receptor antibody forms a conjugate with a cytotoxin. 23. The kit of claim 22, wherein the cytotoxin is selected from the group consisting of saporin, diphtheria toxin, ricin, and herpes thymidine kinase. The kit according to claim 19, which is contained in the pharmaceutical composition so that the local concentration at the site where the oncostatin M antagonist is administered is in an amount of 0.1 to 100 mg / ml. 20. The kit of claim 19, further comprising an anti-oncostatin M antagonist antibody conjugated with a cytotoxin. 21. The kit of claim 19, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 21. The kit of claim 19, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. 28. further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. The kit according to 1. A pharmaceutical composition for treating pain, the composition comprising:
(A) a gene transfer vector comprising a control region of an oncostatin M receptor β chain gene and a cytotoxic gene operably linked to the control region; and (b) a pharmaceutically acceptable carrier. A pharmaceutical composition for treating pain, comprising: 30. The pharmaceutical composition according to claim 29, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene. The pharmaceutical composition according to claim 30, wherein the sequence of the promoter is a sequence consisting of the sequence set forth in SEQ ID NO: 1. 30. The pharmaceutical composition of claim 29, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus. 33. The pharmaceutical composition of claim 32, wherein the gene transfer vector is derived from an adenovirus, an adeno-associated virus, a retrovirus, or a lentivirus. The pharmaceutical composition according to claim 32, wherein the gene transfer vector is derived from a retrovirus. 30. The pharmaceutical composition of claim 29, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, ricin, and herpes thymidine kinase. 30. The pharmaceutical composition of claim 29, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 30. The pharmaceutical composition of claim 29, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. 30. Further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. A pharmaceutical composition according to 1. Use of a gene transfer vector in the manufacture of a medicament for treating pain, the vector comprising a control region of an oncostatin M receptor β chain gene and a cytotoxic gene operably linked to the control region ,use. 40. Use according to claim 39, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene. The use according to claim 40, wherein the sequence of the promoter is a sequence consisting of the sequence set forth in SEQ ID NO: 1. 40. Use according to claim 39, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus. 43. Use according to claim 42, wherein the gene transfer vector is derived from adenovirus, adeno-associated virus, retrovirus or lentivirus. 43. Use according to claim 42, wherein the gene transfer vector is derived from a retrovirus. 40. Use according to claim 39, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, ricin, and herpes thymidine kinase. 40. Use according to claim 39, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 30. A kit for treating pain comprising the pharmaceutical composition of claim 29. 48. The kit according to claim 47, wherein the control region of the oncostatin M receptor β chain gene is derived from the promoter region of the gene. The kit according to claim 48, wherein the sequence of the promoter is a sequence consisting of the sequence set forth in SEQ ID NO: 1. 48. The kit of claim 47, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus, and lentivirus. 51. The kit of claim 50, wherein the gene transfer vector is derived from an adenovirus, an adeno-associated virus, a retrovirus, or a lentivirus. 51. The kit according to claim 50, wherein the gene transfer vector is derived from a retrovirus. 48. The kit of claim 47, wherein the cytotoxic gene is derived from a gene selected from the group consisting of: diphtheria toxin, ricin, and herpes thymidine kinase. 48. The kit of claim 47, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 48. The kit of claim 47, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. 48. Further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. The kit according to 1. A pharmaceutical composition for treating pain comprising at least one of oncostatin M or oncostatin M homolog and a pharmaceutically acceptable carrier,
Here, an oncostatin M homolog is a polypeptide that is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid set forth in SEQ ID NO: 4, 6 or 8, and has oncostatin M activity, wherein Oncostatin M activity is the activity of promoting the proliferation of pluripotent hematopoietic progenitor cells, the activity of promoting the formation of endothelial cell clusters, the activity of promoting differentiation of hepatocytes, the activity of promoting the proliferation of newborn Sertoli cells, and the induction of production of acute phase proteins from hepatocytes A pharmaceutical composition which is at least one activity selected from the group consisting of an activity, a TIMP1 expression-inducing activity, and a liver regeneration promoting activity. 58. The pharmaceutical composition according to claim 57, wherein Oncostatin M or Oncostatin M homolog is contained such that the local concentration at the site of administration is in an amount of 0.1-100 mg / ml. 58. The pharmaceutical composition of claim 57, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 58. The pharmaceutical composition of claim 57, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. 58. Further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. A pharmaceutical composition according to 1.   58. The pharmaceutical composition of claim 57, formulated to be suitable for subcutaneous or intramuscular administration. 58. The pharmaceutical composition of claim 57, wherein the pain is selected from the group consisting of: inflammatory pain, neuropathic pain, and cancer pain. 58. The pharmaceutical composition of claim 57, further comprising at least one of a non-steroidal anti-inflammatory analgesic and a narcotic. 58. Further comprising at least one of indomethacin, aspirin, sodium salicylate, acetaminophen, morphine, fentanyl, alfentanil, sufentanil, remifentanil, and [D-Ala2, MePhe4, Gly-ol5] -enkephalin. A pharmaceutical composition according to 1. Use of Oncostatin M or Oncostatin M homologue in the manufacture of a medicament for treating pain. 58. A kit for treating pain comprising the pharmaceutical composition of claim 57. A pharmaceutical composition for treating pain, the composition comprising:
(A) a gene transfer vector comprising an oncostatin M gene or an oncostatin M homolog gene,
Here, an oncostatin M homolog is a polypeptide that is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid set forth in SEQ ID NO: 4, 6 or 8, and has oncostatin M activity, wherein Oncostatin M activity is the activity of promoting the proliferation of pluripotent hematopoietic progenitor cells, the activity of promoting the formation of endothelial cell clusters, the activity of promoting differentiation of hepatocytes, the activity of promoting the proliferation of newborn Sertoli cells, and the induction of production of acute phase proteins from hepatocytes A gene transfer vector which is at least one activity selected from the group consisting of activity, TIMP1 expression-inducing activity, and liver regeneration promoting activity,
And (b) a pharmaceutical composition for treating pain comprising a pharmaceutically acceptable carrier. 69. The pharmaceutical composition of claim 68, wherein the gene transfer vector is derived from a virus selected from the group consisting of: retrovirus, Sendai virus, adenovirus, adeno-associated virus and lentivirus. 70. The pharmaceutical composition of claim 69, wherein the gene transfer vector is derived from an adenovirus, an adeno-associated virus, a retrovirus, or a lentivirus. The pharmaceutical composition according to claim 70, wherein the gene transfer vector is derived from a retrovirus.