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WO2008044627A1 - Agent prophylactique/thérapeutique destiné aux troubles hépatiques utilisant un baculovirus - Google Patents

Agent prophylactique/thérapeutique destiné aux troubles hépatiques utilisant un baculovirus Download PDF

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Publication number
WO2008044627A1
WO2008044627A1 PCT/JP2007/069554 JP2007069554W WO2008044627A1 WO 2008044627 A1 WO2008044627 A1 WO 2008044627A1 JP 2007069554 W JP2007069554 W JP 2007069554W WO 2008044627 A1 WO2008044627 A1 WO 2008044627A1
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baculovirus
liver
prevention
administration
treatment
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Japanese (ja)
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Hiroshi Takaku
Kahoko Hashimoto
Konomi Nishibe
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BACHTECH Inc
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BACHTECH Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention provides a prophylactic / therapeutic agent for liver disorders including baculovirus.
  • Hepatitis is one of lifestyle-related diseases in Japan. causes of hepatitis include hepatitis virus, alcohol, drugs and poisons. When inflammation occurs in the liver, it progresses to acute hepatitis, chronic hepatitis, cirrhosis, and liver cancer. Acute hepatitis is generally a disease with a good prognosis. In some cases, it progresses to fulminant hepatitis, resulting in death. If hepatitis continues from acute hepatitis for more than 6 months, it progresses to chronic hepatitis. Chronic hepatitis often lacks the subjective symptoms of severe liver dysfunction, so it does not result in death unless an acute liver failure occurs.
  • Cirrhosis has a very high risk of shifting to cirrhosis and leading to the development of hepatocellular carcinoma.
  • Cirrhosis is not a single independent disease, but rather an end image after a chronic course of hepatic lesions caused by various causes has not healed.
  • a characteristic of cirrhosis is fibrosis in the liver. Collage caused by cells in normal liver The balance between monogen synthesis and protease degradation is balanced (Non-patent literature)
  • Non-Patent Document 2 Methods such as hepatocyte transplantation and gene therapy are currently being studied in the treatment of fibrosis! /, The power is still good! /, Results have been obtained! /, Na! /.
  • baculovirus is an entomopathogenic virus that infects insects such as Lepidoptera, Hymenoptera and Diptera.
  • Baculovirus has a circular double-stranded DNA of about 130 kbp as a gene.
  • Autographa californica is a virus that makes inclusion bodies called polyhedra in the nucleus of infected cells in large quantities to reach 40 to 50% of the total cellular protein (Autographa californica).
  • Nucle ar Polyhedrosis Virus: AcNPV is known.
  • Inclusion bodies contain many viruses and protect the virus particles.
  • Inclusion bodies are taken up by insect larvae with food, digested by alkaline phosphatase in the intestine of the host, release infectious particles, and infect the intestinal epithelium. The virus then spreads throughout the host, infects other cells, and is released from the cells as the host dies.
  • Baculovirus is used as an agrochemical because of its high host specificity with little environmental impact. Safety is extremely high because there is little concern about phytotoxicity to human livestock and residual toxicity to the harvest.
  • baculovirus has a strong promoter and can synthesize proteins corresponding to 20 to 30% of all intracellular proteins, and is therefore widely used as a protein expression system.
  • research on gene therapy vectors is also progressing. Since baculoviruses do not replicate in mammals, they are safe because they are not incorporated into chromosomes.
  • Recombinant baculoviruses in which the target gene is inserted downstream of a promoter that moves in mammalian cells such as CMV are found in various mammalian cells such as human hepatoma cells, kidney cells, rat hepatoma cells, and primary human hepatocytes. It has been confirmed that the gene can be expressed in
  • Non-Patent Document 3 describes the cultivation of mice and humans due to baculovirus infection. It is described that IFN was derived from a cell line.
  • Non-patent document 4 describes that when baculovirus is added to a mixed culture of human cultured hepatocytes and Kupffer cells, an inflammatory cytokine is expressed.
  • inoculation of noculowinoles into the nasal cavity to activate innate immunity has the effect of prolonging life against the administration of lethal doses of influenza winoles (non-patent literature). 5) and that baculovirus stimulates Toll-like receptor 9 (TLR9), which is important for natural immune activation (Non-patent document 6).
  • TLR9 Toll-like receptor 9
  • Non-Patent Document 1 Nakamura et al., Latest Medicine, Vol. 55, No. 8, pp. 33-40 (2000)
  • Non-Patent Document 2 Bataller et al., J Clin Invest 115, 209-218 (2005)
  • Non-patent document 3 Gronowski et al., J Virol 73, 9944-9951 (1999)
  • Non-Patent Document 4 Beck et al., Gene Ther 7, 1274-1283 (2000)
  • Non-Patent Document 5 Abe et al., J Immunol 171, 1133-1139 (2003)
  • Non-Patent Document 6 Abe et al., J Virol 79, 2847-2858 (2005)
  • An object of the present invention is to provide a novel means capable of preventing and treating liver damage and / or suppressing liver fibrosis.
  • HGF which is a liver regeneration factor
  • IFNs which are hepatic fibrosis-inhibiting factors
  • liver fibrosis including baculovirus
  • liver disorder Prevention of liver disorder 'Prevention of the disease comprising a baculovirus inserted in a manner capable of expressing a nucleic acid encoding a therapeutically effective factor, and applied to a subject suffering from liver disorder ⁇ Therapeutic drugs;
  • Concomitant drugs including baculoviruses, and preventive and therapeutic drugs for liver damage;
  • liver disorder is hepatitis, cirrhosis or liver cancer
  • a method for preventing or treating liver damage in a subject comprising administering an effective amount of baculovirus to the subject;
  • the subject comprising administering an effective amount of a baculovirus inserted in a manner capable of expressing a nucleic acid encoding a factor effective in preventing or treating liver injury to a subject suffering from liver injury How to prevent and treat liver damage in the body.
  • the baculovirus and agent of the present invention can be useful for the prevention and treatment of liver disorders such as hepatitis, cirrhosis and liver cancer, and / or suppression of liver fibrosis.
  • FIG. 1 is a diagram showing the administration schedule of DMN and baculovirus, LPS, or physiological saline.
  • DMN (10 mg / kg) was administered to BALB / c mice, followed by intraperitoneal administration of baculovirus (10 3 , 10 5 , 10 7 pfu), LPS ⁇ S g) and physiological saline.
  • BV Baculovirus (same below).
  • FIG. 2 is a graph showing GOT values in mice treated with baculovirus. GOT values in serum collected from each treatment group were measured. The DMN and physiological saline administration groups showed high values, while the baculovirus administration group showed significantly low values. There was no significant difference in the titer of noculovirus.
  • Statistical analysis Software StatView
  • FIG. 3 is a graph showing GPT values in baculovirus-administered mice. GPT values in serum collected from each treatment group were measured. The DMN and saline administration groups showed high values, while the baculovirus administration group showed significantly low values. There was no significant difference between the titers of baculovirus.
  • Statistical analysis Software StatView Ver 4.11, ANOVA, Scheffe's F test
  • FIG. 4 is a graph showing the amount of collagen in mice treated with baculovirus.
  • the amount of collagen in the liver of each administration group was measured.
  • the DMN and saline administration groups showed high values, while the baculovirus administration group showed significantly low values. There was no significant difference in potency due to baculovirus.
  • Statistical analysis Software Stat Vie w Ver4. 11, ANOVA, Scheffe's F test
  • FIG. 5 is a diagram showing a baculovirus or LPS administration schedule.
  • Baculovirus (10 7 pfu) and LPS (75; ⁇ ) were intraperitoneally administered to BALB / c mice. At 12, 12, 24, and 48 hours after administration, dissection was performed, and intestinal lymph nodes, intraperitoneal cells, spleen, liver, and blood were collected. The LPS administration group was dissected 2 hours after administration.
  • the present invention provides an agent containing a baculovirus.
  • baculovirus vector More than 500 baculoviruses have been isolated from diseased insects in the field, and there are 603 registered as temporary species.
  • the baculoviridae is a nuclear polyhedrosis virus ( Nuclear polyhedrosis virus (NPV) and granulovirus (GV) are broadly classified.
  • NPV nuclear polyhedrosis virus
  • GV granulovirus
  • NPV is preferably used as the baculovirus vector.
  • the NPV that can be used in the present invention includes, for example, Aut ographa californica NPV (AcNPV) isolated from a kind of cocoon, Bombvx mori NPV (BmNPV) derived from silkworm, Orgyia pseudotsugata NPV (OpNPV) derived from silkworm, maimaiga AcNPV is preferred among the strengths of Lymantria dispar NPV (LdNPV) derived from Spodoptera exigua NPV (SeNPV) derived from Shirochimojoto.
  • AuNPV Aut ographa californica NPV
  • BmNPV Bombvx mori NPV
  • OpNPV pseudotsugata NPV
  • LdNPV Lymantria dispar NPV
  • SeNPV Spodoptera exigua NPV
  • the baculovirus may be inserted in such a manner that a nucleic acid encoding a factor effective for the prevention and treatment of a predetermined disease can be expressed.
  • a nucleic acid include a nucleic acid encoding a factor (peptide, protein) effective for prevention / treatment of a predetermined disease or a nucleic acid itself.
  • the nucleic acid itself include an antisense nucleic acid, a ribozyme, an RNAi-derived nucleic acid (eg, siRNA), an abutama, and a decoy nucleic acid.
  • the baculovirus may also contain factors that improve gene expression efficiency. Examples of factors that improve gene expression efficiency include VSV-G, RVG (rabies virus G glycoprotein), and MHV (mouse hepatitis virus).
  • the present invention also provides such a baculovirus.
  • a baculovirus When a baculovirus contains a nucleic acid encoding a factor effective for the prevention and treatment of a given disease, it may contain a promoter operably linked to the nucleic acid.
  • the promoter can be appropriately selected depending on whether or not a factor encoded by the nucleic acid or the nucleic acid itself should be expressed. Examples of such promoters include polIII promoters (eg, tRNA promoter, U6 promoter, HI promoter) and mammalian promoters (eg, CMV promoter, CAG promoter, SV40 promoter).
  • a baculovirus containing a nucleic acid encoding a factor effective for prevention / treatment of a predetermined disease can be prepared by a method known per se.
  • a baculovirus containing a nucleic acid encoding the factor incorporates the nucleic acid encoding the factor into a baculovirus transfer vector, and then introduces the recombinant transfer vector and the baculovirus into an insect cell, resulting in homologous recombination. Can be produced.
  • a nucleic acid encoding a factor effective for the treatment and prevention of a predetermined disease there is no particular limitation on the predetermined disease that can be prevented or treated by the factor.
  • the predetermined disease can be, for example, a liver disorder described later or other diseases.
  • noculowinoles contains a nucleic acid that encodes a factor that is effective in the prevention and treatment of liver damage
  • such baculoviruses have a pharmacological effect due to the baculovirus itself and a pharmacological effect due to the factor.
  • Factors effective for the prevention and treatment of liver damage may be, for example, IFN and HGF described below.
  • baculovirus when it contains a nucleic acid that encodes a factor that is effective for the prevention / treatment of diseases other than liver damage, is applied to subjects who have or are at risk of developing the disease and liver damage.
  • Such diseases include, for example, organs or tissues other than the liver (eg, brain, viscera, kidney, liver, gonad, thyroid, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, stomach, large intestine, small intestine). ), Diseases in the heart, thymus, spleen, lymph nodes, peripheral blood, bone marrow), infections (eg, viral diseases such as AIDS, influenza), cancer, autoimmune diseases, neuroimmune diseases, and / or! Includes the diseases described below.
  • the subject to which baculovirus is administered in the present invention is usually a mammal.
  • Mammals include, for example, laboratory animals such as rodents such as mice, rats, mice, musters, and guinea pigs, and domestic animals such as pigs, rabbits, goats, horses, hidges, minks, Examples include pets such as dogs and cats, primates such as humans, sanorets, lizards, marmosets, orangutans and chimpanzees.
  • the present invention also provides a combination of a baculovirus and a prophylactic and therapeutic agent for liver damage
  • the drug for preventing or treating liver damage is not particularly limited as long as it has an effective pharmacological activity for liver damage.
  • a low molecular weight organic compound eg, non-peptidic compound, non-proteinaceous
  • Compounds, non-nucleic acid substances), and factors eg, peptides, proteins, nucleic acids
  • the combination of the present invention can be, for example, a concomitant drug.
  • the concomitant drug of the present invention may be a combination of noculowinoles and a prophylactic / therapeutic agent for liver damage.
  • the combination of the present invention may also be a kit.
  • the kit of the present invention comprises a noculovirus, And prevention / treatment of liver disorders may be included individually (ie, in an unmixed manner).
  • the agent or combination of the present invention may contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to baculovirus.
  • pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose. , Polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate, etc.
  • Agent Magnesium stearate, air mouth gel, talc, sodium lauryl sulfate, lubricant, citrate, menthol, glycyrrhizin ammonium salt, glycine, orange powder
  • Preservatives such as sodium, sodium bisulfite, methylparaben, propylparaben, stabilizers such as citrate, sodium citrate, acetic acid, suspension agents such as methylcellulose, polybutylpyrrolidone, aluminum stearate, surfactants, etc.
  • Dispersants water, physiological saline, diluents such as orange juice, cocoa butter, polyethylene glycol, base waxes such as white kerosene, and the like.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile formulations, which include antioxidants, buffers , Including antibacterial agents, tonicity agents, etc.!
  • aqueous and non-aqueous sterile suspensions can also be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the preparation can be enclosed in a unit dose or multiple doses like ampoules and vials.
  • the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state where it may be dissolved or suspended in a suitable sterile vehicle immediately before use.
  • the dose of baculovirus may vary depending on the activity and type of the active ingredient, the mode of administration, the severity of the disease, the animal species to be administered, the drug acceptability of the administration subject, body weight, age, etc. Can be set.
  • the agent or combination of the present invention may be useful as a medicament.
  • prevention of liver damage Can be useful for treatment.
  • liver disorders that can be prevented and treated with baculovirus include hepatitis (acute hepatitis, chronic hepatitis), cirrhosis, and liver cancer.
  • hepatitis include those caused by hepatitis virus, alcohol, or drug 'toxins.
  • the agent or combination of the present invention may also be useful for the prevention and treatment of diseases other than liver disorders.
  • the agent of the present invention is useful for the treatment of diseases in which induction of IFN is desired in the living body.
  • the disease for which induction of IFN is desired is not particularly limited as long as it is a disease that can be prevented or treated by the pharmacological activity of IFN, and includes, for example, cancer (including leukemia, malignant lymphoma, sarcoma, and the like). Severe acute respiratory syndrome (SAR S), chronic obstructive pulmonary disease (COPD), rabies, neurological disease, gastroduodenal disease.
  • SAR S Severe acute respiratory syndrome
  • COPD chronic obstructive pulmonary disease
  • rabies neurological disease
  • gastroduodenal disease gastroduodenal disease.
  • the agent of the present invention may be useful for treatment of a disease for which induction of HGF is desired in the living body.
  • the disease for which induction of HGF is desired is not particularly limited as long as it is a disease that can be prevented or treated by the pharmacological activity of HGF, such as neurological disease, obstructive arteriosclerosis, diabetic peripheral neuropathy, angiogenesis, Cancer, alopecia (eg male alopecia), inflammatory bowel disease.
  • the agent of the present invention may be useful for the treatment of a disease for which suppression of TGF- / 3 expression in a living body is desired.
  • the disease for which suppression of TGF-3 expression is desired is not particularly limited as long as the disease can be exacerbated by TGF- ⁇ , and examples thereof include rheumatism, allergy, and oral cancer.
  • the agent or the combination of the present invention may be applied (administered) to a subject having or at risk of developing one or more diseases described above, for example.
  • Example 1 Liver fibrosis model mouse tree
  • DNN Dimethylnitrosamine
  • liver fibrosis model by repeatedly administering DMN every other day and causing chronic hepatitis-like symptoms.
  • BALB / c mice female, 7 weeks old
  • DMN 10 mg / kg
  • This administration condition was continued for 1 to 5 weeks (group).
  • Group 5 This administration condition follows Lasts 5 weeks).
  • Each week, dissection was performed on the day of DMN administration for that week, and blood and liver were removed. The results are as follows.
  • mice Body weight loss was similar in each group of mice, with significant weight loss observed on days 4 and 5 after DMN administration at the beginning of each week. Mice administered with saline showed the same increase or decrease in body weight as mice not administered.
  • GOT Glutamic acid-Oxaloacetic Transaminase
  • GPT glutamic acid-pyruvate transaminase
  • liver fibrosis is induced when collagen accumulates significantly in the liver. Therefore, we examined whether liver fibrosis actually occurred by quantifying collagen in the liver. In mice treated with DMN for 3 or 4 weeks, the DMN administration group showed a high collagen content.
  • AcNPV Autographa californica NPV
  • NPV nuclear polyhedrosis virus
  • Insect-derived Sf-9 cells were used as cultured cells. Sf-9 cells were seeded in a culture dish ( ⁇ 10 cm). When Sf-9 cells became 80% confluent, the virus solution was added dropwise. Thereafter, the cells were cultured at 27 ° C. for 3 to 4 days to propagate the virus. This operation was continued until the culture supernatant finally became 1-2 L.
  • the collected culture supernatant was centrifuged at 1500 rpm for 10 minutes to separate into cells and virus solution.
  • the virus solution was centrifuged at 8000 rpm, 4 ° C for 12 hours to remove the supernatant, and the virus mass was suspended in 1 X PBS (1). This solution was purified by sucrose gradient.
  • the titer of baculovirus was determined by plaque assay.
  • Sf-9 cells were seeded in a culture dish ( ⁇ 60 mm) to 80% confluence and allowed to stand for 1 hour to adhere. After attachment, the cells were washed with serum-free Sf-900II medium (Invitrogen), and 4001 serum-free medium was added.
  • the virus solution was serially diluted with serum-free Sf-900 ⁇ medium to prepare a virus dilution. 100 ml of this virus solution was added dropwise to Sf-9 cells and adsorbed and infected at room temperature for 90 minutes. During adsorptive infection, virus solution was applied to Sf-9 cells every 10 minutes.
  • Virus titer [ ⁇ (dilution rate) X (number of plaques) ⁇ / (number of dilution rate)] X 10 (I)
  • Example 3 Treatment of liver fibrosis by baculovirus administration
  • Example 1 From Example 1, it was found that for the construction of liver fibrosis model mice, DMN was administered twice a week and repeated for 3 weeks. Therefore, in this example, baculovirus was administered two days after the peak of weight loss after the second DMN administration weekly, and the effect of treating liver fibrosis was examined.
  • a liver fibrosis model was constructed by administering DMN (10 mg / kg) twice a day into the abdominal cavity of BALB / c mice (female 7 weeks old) and continuing this for 3 weeks.
  • DMN 10 mg / kg
  • mice on the day of administration were dissected and used as the standard for each measurement.
  • Baculovirus (10 3 , 10 5 , 10/200 1 / animal) and LPS (75 1 / animal) and physiological saline (200 1 / animal) were administered twice abdominally 2 days after the final administration of DMN. LPS was used as a control.
  • Figure 1 shows the dosing schedule. Serum was collected and GOT'GPT was measured. Collagen was extracted from the liver using 0.5M acetate buffer, and the amount of collagen was measured. In addition, after immobilizing a part of the liver using 20% formalin solution, perform Masson's trichrome staining and Hematoxylin-eosin (HE) staining to analyze collagen accumulation and cell status! / Observed under a microscope.
  • HE Hematoxylin-eosin
  • mice After 3 weeks of DMN administration, body weight changes were measured when baculovirus, saline and LPS were administered. In the physiological saline and LPS administration group, the mice showed fuzz, but in the baculovirus administration group, no fuzz was observed even though the body weight decreased.
  • Tissue damage was analyzed by measuring GOT'GPT in serum collected from the blood of each treatment group (Figs. 2 and 3). Saline administration did not reduce the effects of DMN on the liver, but both GOT and GPT showed high values, but baculovirus administration showed no significant difference between each concentration Compared with saline administration Significantly lower Indicated.
  • liver collagen which is an index of liver fibrosis, caused by baculovirus administration
  • the amount of collagen in the liver was measured (Fig. 4).
  • the administration of physiological saline showed a high value without affecting the collagen accumulation by DMN.
  • the curovirus administration did not show a significant difference between the concentrations, but the value was significantly lower than that of physiological saline.
  • Masson's trichrome staining and pathological analysis dyes nuclei with iron to matoxylin, followed by small dye molecules with high diffusion rate (acid fuchsin, ponsoxylysine) penetrating into the reticulopoies of cells, followed by large dyes with low diffusion rate.
  • This is a staining method in which molecules (anilin blue) enter the crude structure of collagen fibers and dye them in blue. Collagen fibers are stained blue, cytoplasm is pink, and cell nuclei are stained purple. Improvement of intra-sinusoidal fibril formation by DMN was not observed with saline administration, and fibrosis was reduced with baculovirus administration, indicating a correlation with collagen content.
  • HE staining is the most common staining method and is mainly used for cytopathological diagnosis. Hematoxylin solution stains nuclei 'ribosomes etc. in blue-blue, and eosin solution stains cytoplasm' fibers' erythrocytes in red. Significant lobular central cannon degeneration was observed with DMN administration, and no improvement in degeneration was observed with saline administration. However, with baculovirus administration, denaturation was reduced in proportion to the dose (virus titer).
  • Example 4 Biological response of mice by baculovirus administration
  • Example 3 it was shown that hepatic fibrosis was improved by baculovirus administration, and in particular, a high improvement effect was obtained with 10 7 pfu of baculovirus. Since the gene expression of site force-in due to viral infection is expressed in a short time after infection, baculovirus is intraperitoneally injected into mice. It was confirmed over time what kind of cells were infected and what kind of site force was expressed.
  • baculovirus In order to confirm which cells are infected by intraperitoneal administration of baculovirus, DNA was extracted from peritoneal lymph nodes (intestinal lymph nodes), intraperitoneal cells and spleen force, and PCR was used to detect the Vp39 gene. It was. Invasion of baculovirus was confirmed in each organization. Presence of both baculovirus doses was confirmed with both single and double doses. In particular, the presence of baculovirus DNA could be confirmed even on the fourth day in intraperitoneal cells and spleen. In addition, since it exists in intraperitoneal cells, it is considered that baculovirus is infected with phagocytic cells with complement receptors and carried to tissues that induce immune responses.
  • vp39 gene expression was observed in the spleen.
  • the spleen also called secondary lymphoid tissue, is a collection of immune cells. When a pathogen invades, it is an important organ for inducing a strong immune response in the early stages of infection. Therefore, the presence of baculovirus in the spleen activates the immune response.
  • the expression of IFNs by baculovirus infection has been reported (Gronowski et al., J Virol 73, 9944-9951 (1999)), so IFNs gene expression in each tissue was confirmed using RT-PCR. did.
  • IFN- ⁇ Gene expression of IFN- ⁇ was confirmed in the intestinal lymph node, intraperitoneal cells and spleen, and gene expression of IFN- ⁇ was confirmed in intraperitoneal cells and spleen. The expression of the IFN- ⁇ gene was unidentifiable. In the intestinal lymph nodes, IFN— gene expression was confirmed after two doses of baculovirus, so immunity was established and memorized by the first dose, and the second round of baculovirus progression. It is thought that the IFN- / 3 gene was expressed in order to eliminate the virus. IFN-7 was an intraperitoneal cell, and its gene expression was confirmed 12 hours after the second baculovirus administration.
  • IFNs, HGF, and TGF- ⁇ mRNA expression in the liver were examined by RT-PCR.
  • the gene expression of IFN-0 was confirmed at 12, 24 and 48 hours after the second dose of baculovirus.
  • HGF a liver regeneration factor
  • TGF- ⁇ gene expression decreased after 12 hours. This suggests that the HGF protein strongly induced by baculovirus administration suppresses TGF- ⁇ gene expression.
  • the HGF gene expression decreased after 12 hours because HGF was overexpressed by baculovirus, and after 48 hours it returned to the same level of expression as BAL B / c mice!
  • baculovirus can be effective in suppressing liver fibrosis and improving liver damage.
  • the baculovirus and agent of the present invention can be useful for the prevention and treatment of liver disorders such as hepatitis, cirrhosis, and liver cancer, and / or suppression of liver fibrosis.

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Abstract

La présente invention concerne les éléments suivants : un agent prophylactique/thérapeutique destiné aux troubles hépatiques, qui comprend un baculovirus ; un inhibiteur de fibrose du foie, qui comprend un baculovirus ; un baculovirus présentant, inséré dedans de manière opérable, un acide nucléique codant pour un facteur qui est efficace dans la prévention/le traitement d'un trouble hépatique ; un agent prophylactique/thérapeutique destiné à une maladie autre qu'un trouble hépatique, qui comprend un baculovirus codant pour un facteur efficace dans la prévention/le traitement de la maladie et qui est appliqué à un sujet présentant lesdits troubles hépatiques ; un kit ou un agent combiné comprenant le baculovirus ou l'agent prophylactique/thérapeutique destiné à un trouble hépatique ; et d'autres éléments.
PCT/JP2007/069554 2006-10-06 2007-10-05 Agent prophylactique/thérapeutique destiné aux troubles hépatiques utilisant un baculovirus Ceased WO2008044627A1 (fr)

Priority Applications (1)

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JP2008538696A JPWO2008044627A1 (ja) 2006-10-06 2007-10-05 バキュロウイルスを利用した肝障害の予防・治療剤

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JP2006275715 2006-10-06
JP2006-275715 2006-10-06

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WO2008044627A1 true WO2008044627A1 (fr) 2008-04-17

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PCT/JP2007/069554 Ceased WO2008044627A1 (fr) 2006-10-06 2007-10-05 Agent prophylactique/thérapeutique destiné aux troubles hépatiques utilisant un baculovirus

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JP (1) JPWO2008044627A1 (fr)
WO (1) WO2008044627A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017141942A1 (fr) * 2016-02-16 2017-08-24 国立大学法人大阪大学 Composition médicinale pour le traitement de la fibrose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NISHIBE Y. ET AL.: "Baculovirus Kansen Men'eki ni yoru Kan Sen'ika Chiryo", PROCEEDINGS OF THE JAPANESE SOCIETY FOR IMMUNOLOGY, vol. 34, 5 November 2004 (2004-11-05), pages 63, XP003022200 *
NISHIBE Y. ET AL.: "Baculovirus ni yoru Kan Sen'ika no Kaizen", PROCEEDINGS OF THE JAPANESE SOCIETY FOR IMMUNOLOGY, vol. 35, 15 November 2005 (2005-11-15), pages 55, XP003022301 *
SUZUKI H. ET AL.: "Suppression of HCV RNA replication by baculovirus-mediated shRNA expression", NUCLEIC ACIDS SYMPOSIUM SERIES, no. 49, 2005, pages 339 - 340, XP003022199 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017141942A1 (fr) * 2016-02-16 2017-08-24 国立大学法人大阪大学 Composition médicinale pour le traitement de la fibrose
JPWO2017141942A1 (ja) * 2016-02-16 2018-12-20 国立大学法人大阪大学 線維化を治療するための医薬組成物
CN109152804A (zh) * 2016-02-16 2019-01-04 国立大学法人大阪大学 用于治疗纤维化的药物组合物
US11077157B2 (en) 2016-02-16 2021-08-03 Osaka University Medicinal composition for treating fibrosis

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