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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates to a therapeutic agent for nervous system tumors such as neuroblastoma.
• Neuroblastoma (neuroblastoma) is a childhood malignancy of ganglion neurons that usually begins by the age of three and often has unfortunate consequences. Neuroblastoma is the most frequent of solid tumors in children and cures spontaneously in many cases, but is accompanied by amplification of N-myc and many malignant cases. Because patients are children, there is no effective therapeutic drug, and the development of new treatments is desired. Disclosure of the invention
• the present inventors focused on Hu protein, an RNA-binding protein that specifically expresses in differentiated neurons, and introduced Hu protein gene into SH-SY cells, which are neuroblastomas, to overexpress Hu protein. When expressed, they found that they induced apoptosis and markedly stopped cell proliferation, and completed the present invention.
• the present invention encodes a Hu protein, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted, added or inserted in the amino acid sequence of the Hu protein, or encodes these amino acid sequences. It is intended to provide a therapeutic agent for a nervous system tumor containing a gene as an active ingredient.
• the present invention also relates to a Hu protein, a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted, added or inserted into the amino acid sequence of Hu protein, or a gene encoding these amino acid sequences.
• a Hu protein a polypeptide having an amino acid sequence in which one or more amino acids have been substituted, deleted, added or inserted into the amino acid sequence of Hu protein, or a gene encoding these amino acid sequences.
• the present invention provides a Hu protein, a polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted into the amino acid sequence of Hu protein, or an effective amount of a gene encoding these amino acid sequences. And a method for treating a nervous system tumor characterized by administering
• FIG. 1 shows micrographs (a and d), immunostaining results (b and e), and hoechst staining results (c and f) of SH-SY5Y cells into which HuB was introduced.
• FIG. 2 is a diagram showing the TUNEL-positive rate (%) [TUNEL-positive cells / FLAG (or Myc) -positive cells] of SH-SY5Y cells into which HuB was introduced and controls.
• Figure 3 shows the results of immunostaining of SH-SY5Y cells 48 hours after introduction of HuB using anti-BrdU antibody and anti-FLAG antibody or anti-Myc antibody (a shows staining with BrdU antibody, b shows FLAG Staining with antibodies).
• FIG. 4 is a diagram showing the BrdU positive rate between HuB-introduced cells and GFP (control).
• FIG. 5 shows the results of immunostaining of Be1-2 antibody of HuB-introduced SH-SY5Y cells (a shows staining with Bd-2 antibody, and b shows staining with FLAG antibody).
• FIG. 6 is a diagram showing the results of immunoblot with the p27 antibody.
• FIG. 7 shows the subcloning strategies of UTR-1, UTR_2, and UTR-3 of the human bd-2 gene.
• FIG. 8 shows a strategy for point mutation in RRM2.
• FIG. 9 is a diagram showing the binding of bd-2 mRNA 3 ′ UTR-1, 2, and 3 to HuB.
• the Hu protein which is an active ingredient of the medicament of the present invention, is a protein identified as an antigen recognized by an autoantibody appearing during neuropathy associated with small cell lung cancer.
• Hu The protein is an RNA-binding protein that shows specific expression in differentiated neurons, and binds to the AU-rich element (ARE) located on the 3 'UTR side of the target mRNA to regulate the expression of the target gene product. It is known to have a function of regulating at the post-transcriptional level, but its effect on neuroblastoma is not known at all.
• Hu protein can be isolated from the cell in which it is present, but since the gene encoding the Hu protein has already been cloned, DNA recombination technology, that is, an expression vector prepared using the gene, can be used. May be prepared using transformed cells.
• the Hu protein may be the protein itself expressed in differentiated neurons, but may have a partially modified amino acid sequence as long as it has similar properties.
• a polypeptide having an amino acid sequence in which one or more of the amino acid sequences of the Hu protein are substituted, deleted, added, or inserted may be used.
• the degree of these substitutions, deletions, additions or insertions and their positions are not particularly limited as long as the modified amino acid sequence has the same properties as the Hu protein.
• the modification preferably has a homology of usually 80% or more, particularly preferably 90% or more.
• These modified polypeptides can also be prepared by a DNA recombination technique like the Hu protein.
• gene therapy in which a gene encoding the Hu protein or the modified polypeptide is administered, and the protein or the modified polypeptide is produced in the body may be employed. Since these genes are also cloned in a known manner, it is preferable to use them.
• Hu protein or Hu protein gene is useful as an agent for treating nervous system tumors such as neuroblastoma.
• the expression of p27 is increased in the cell growth inhibitory effect of the Hu protein in SH-SY cells, and the suppression of Bel-2 expression is involved in the apoptosis-inducing effect.
• various active forms can be obtained by adding pharmaceutically acceptable carriers to the active ingredient.
• an injectable preparation is preferable.
• Pharmaceutically acceptable carriers include distilled water, solubilizers, stabilizers, emulsifiers, buffers and the like. The dosage of these drugs varies depending on the disease, gender, body weight, etc., but will be about 0.1 g to 10 mg / day in terms of Hu protein mass or Hu protein gene amount.
• Plasmid (Akamai su et al. PNAS 1999) incorporated into pCXN2, a vector for forced expression of mammalian cells with a FLAG-tag, was added to Lipofectamine.
• the TUNEL positive rate of the transfected cells was calculated by the TUNEL method.
• the cells transfected with HuB about 4 times as many cells as the control (GFP-Myc) were TUNEL-positive, and apoptosis was enhanced (Fig. 2).
• G418 was added, the transfected cells were selected, and observation was performed for 7 days. However, cells that further expanded the neurites and differentiated or cells that continued to proliferate and formed colonies could not be confirmed.
• the SH-SY5Y cells into which HuB had been introduced were introduced from 36 hours to 12 hours after the introduction, with the addition of prodoxydziridine (BrdU) to the medium to label the cells in the S phase. 48 hours after transfection, immunostaining was performed using anti-BrdU antibody and anti-FLAG or Myc antibody (Fig. 3a, b), and the positive rate of BrdU in the transfected cells was calculated (Fig. 4).
• SHB-SY5Y cells into which HuB had been introduced showed about a 50% decrease in BrdU uptake as compared to the control. That is, overexpression of HuB stopped cell growth of SH-SY5Y cells.
• Bcl-2 is a differentiation marker that is known to increase with the differentiation of SH-SY5Y cells. Twenty-four hours after the introduction of HuB-transfected SH-SY5Y cells, immunostaining was performed with the antibody Be1-2. The results are shown in FIGS. 5a and b. In HuB-introduced cells (FLAG-positive cells), the expression of Bel-2 indicated by the white arrow was reduced. Dotted lines indicate the perimeter of individual cells. Next, SH-SY5Y cells transfected with HuB, HuC, and control (GFP-Myc) were subjected to immunoblot with a P27 antibody that has been reported to bind Hud protein to Bd-2 (Fig. 6). ).
• the human bcl-2 gene has a long UTR portion of 5.5 kb in length, in which AU-rich
• ARE element
• pGEX-HuB and HuB-R2mt were expressed in Escherichia coli BL21, and purified on glutathione sepharose. 200 ng of the purified protein was mixed with be 2 mRNA 3 ′ UTR-1, 2, 3 labeled with 32 P-UTP, and subjected to UV-crosslin for 1 minute with Stratlinker. 12. The sample was electrophoresed on a 5% -SDS-PAGE gel and detected by BAS-5000 (Fig. 9).
• HuB showed binding to UTR-1 and UTR-2, but not UTR-3 without the AU-rich element. HuB-R2 showed no binding to any of UTR1-3.
• HuB was found to bind to bd-2 mRNA containing the AU-rich portion, and that binding was lost when a point mutation was introduced into RRM2.
• the present invention has provided a new method for treating neuroblastoma, which has been difficult to treat.

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• 2001
  • 2001-05-24 JP JP2001155237A patent/JP2002348254A/ja active Pending
  • 2001-10-03 CA CA002446510A patent/CA2446510A1/en not_active Abandoned
  • 2001-10-03 WO PCT/JP2001/008701 patent/WO2002094306A1/ja not_active Ceased
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• 2007
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