WO2008044627A1

WO2008044627A1 - Prophylactic/therapeutic agent for liver disorder utilizing baculovirus

Info

Publication number: WO2008044627A1
Application number: PCT/JP2007/069554
Authority: WO
Inventors: Hiroshi Takaku; Kahoko Hashimoto; Konomi Nishibe
Original assignee: Bachtech, Inc.
Priority date: 2006-10-06
Filing date: 2007-10-05
Publication date: 2008-04-17
Also published as: JPWO2008044627A1

Abstract

Disclosed are: a prophylactic/therapeutic agent for a liver disorder, which comprises a baculovirus; an inhibitor of liver fibrosis, which comprises a baculovirus; a baculovirus having, operably inserted therein, a nucleic acid encoding a factor which is effective for the prevention/treatment of a liver disorder; a prophylactic/therapeutic agent for a disease other than a liver disorder, which comprises a baculovirus encoding a factor effective for the prevention/treatment of the disease and which is applied to a subject having the liver disorder; a combined agent or kit comprising the baculovirus or the prophylactic/therapeutic agent for a liver disorder; and others.

Description

Specification

Prevention and treatment of liver damage using baculovirus

Technical field

[0001] The present invention provides a prophylactic / therapeutic agent for liver disorders including baculovirus.

Background art

[0002] 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. However, chronic hepatitis 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. Liver cirrhosis caused death by liver failure, gastrointestinal hemorrhage, and hepatocellular carcinoma in 20-30 years ago, but recently, hepatocellular carcinoma accounted for 70%, liver failure 20%, digestion Vascular bleeding is 5% and others are 5%. This is due to advances in nutritional therapy, improvement of endoscopic treatment for esophageal varices, and the reduction and death of esophageal varices due to the development and dissemination of antibiotics, diuretics and albumin preparations, etc. There is. Increasing the survival rate of cirrhosis patients inevitably leads to aging of cirrhosis patients, and at the same time, increases the incidence of liver cancer.

[0003] There are two methods for treating cirrhosis: treatment for the purpose of preventing onset or suppressing progression, and treatment for complications. Although the prognosis of life has improved significantly due to the progress of each diagnosis and treatment method, V has not been performed with the therapeutic ability to prevent progression to liver cancer. If the condition can be reduced from cirrhosis to hepatitis level, there is a possibility of complete cure.

[0004] 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)

When damage is applied to Do cells, collagen is secreted and remains as a scar, and much secreted collagen is degraded. However, this balance is imbalanced due to chronic liver damage. Liver fibrosis is the result of hepatic wound healing against repeated injury (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! /.

By the way, baculovirus (BV) 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. As baculoviruses, for example, 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 (polyhedra) 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.

[0006] 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. In addition, 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. Furthermore, 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

[0007] Wild-type baculovirus has been reported to induce immune activation in mammals. For example, 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. In addition, in the laboratory of the present inventors, 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).

[0008] However, it has been reported that baculovirus S can prevent and treat liver damage and / or suppress liver fibrosis! /.

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)

Disclosure of the invention

Problems to be solved by the invention

[0009] An object of the present invention is to provide a novel means capable of preventing and treating liver damage and / or suppressing liver fibrosis.

Means for solving the problem

[0010] As a result of intensive studies, the present inventors have been able to induce HGF, which is a liver regeneration factor, and IFNs, which are hepatic fibrosis-inhibiting factors, in vivo by administration of baculovirus. It has been found that the action of TGF-β, which is a glycation-promoting factor, can be suppressed, and that such effects can prevent and / or treat liver damage and / or can suppress liver fibrosis. It came to complete.

[0011] That is, the present invention is as follows:

[1] Prevention and treatment of liver damage, including baculovirus;

[2] The agent according to [1] above, wherein the baculovirus is a baculovirus vector; [3] The agent according to [1] or [2] above, wherein the liver disorder is hepatitis, cirrhosis or liver cancer;

[4] An inhibitor of liver fibrosis, including baculovirus;

[5] Prevention of hepatic disorder 'Introduced in a manner that allows expression of nucleic acids encoding factors effective for treatment! /, Baculoviruses;

[6] 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;

[7] Concomitant drugs, including baculoviruses, and preventive and therapeutic drugs for liver damage;

[8] Kit containing baculovirus and preventive / therapeutic agent for liver damage;

[9] Prevention of liver damage · Use of baculovirus to produce a therapeutic agent;

[10] The use according to [9] above, wherein the baculovirus is a baculovirus vector;

[11] The use according to [9] or [10] above, wherein the liver disorder is hepatitis, cirrhosis or liver cancer;

[12] Use of baculovirus for producing an inhibitor of liver fibrosis;

[13] Prevention of liver damage applied to a subject suffering from liver damage, in order to produce a nucleic acid encoding a factor effective for the treatment of liver damage prevention to produce a therapeutic agent. Use of existing baculoviruses;

[14] A method for preventing or treating liver damage in a subject, comprising administering an effective amount of baculovirus to the subject;

[15] The method according to [14] above, wherein the baculovirus is a baculovirus vector; [16] the method according to [14] or [15] above, wherein the liver disorder is hepatitis, cirrhosis or liver cancer; Administering to the subject an effective amount of baculovirus, a method for inhibiting liver fibrosis in the subject;

[18] 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 invention's effect

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. Brief Description of Drawings

[0013] 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

Ver4.11, ANOVA, Scheffe 's F test

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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The present invention provides an agent containing a baculovirus.

[0015] 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. In the present invention, 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.

[0016] 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. Examples of such 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. Examples of 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.

[0017] 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).

[0018] 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. For example, 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. [0019] In the case of containing 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. When 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. Synergistic prophylaxis against liver damage. Factors effective for the prevention and treatment of liver damage may be, for example, IFN and HGF described below. On the other hand, 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. Can be done. 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.

[0020] 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.

[0021] The present invention also provides a combination of a baculovirus and a prophylactic and therapeutic agent for liver damage

[0022] The drug for preventing or treating liver damage is not particularly limited as long as it has an effective pharmacological activity for liver damage. For example, a low molecular weight organic compound (eg, non-peptidic compound, non-proteinaceous) Compounds, non-nucleic acid substances), and factors (eg, peptides, proteins, nucleic acids) effective in the prevention and treatment of liver damage.

[0023] 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.

[0024] 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).

[0025] The agent or combination of the present invention may contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to baculovirus. Examples of 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.

[0026] Suitable formulations for parenteral administration (eg, intravenous injection, topical application, etc.) 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. In addition, 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.

[0027] 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.

[0028] The agent or combination of the present invention may be useful as a medicament. For example, prevention of liver damage Can be useful for treatment. Examples of liver disorders that can be prevented and treated with baculovirus include hepatitis (acute hepatitis, chronic hepatitis), cirrhosis, and liver cancer. Examples of hepatitis include those caused by hepatitis virus, alcohol, or drug 'toxins.

[0029] The agent or combination of the present invention may also be useful for the prevention and treatment of diseases other than liver disorders. For example, since baculovirus can induce IFN (eg, IFN-a, IFN-β, IFN-γ) in the living body, the agent of the present invention is useful for the treatment of diseases in which induction of IFN is desired in the living body. Can be useful. 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.

[0030] In addition, since baculovirus can induce HGF, which is a liver regeneration factor in a living body, 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.

[0031] Furthermore, since baculovirus can suppress TGF-β expression in a living body, 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-β, and examples thereof include rheumatism, allergy, and oral cancer.

[0032] 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.

[0033] The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all of them were explicitly stated. It is built in.

[0034] The present invention will be described in more detail with reference to the following examples. It ’s not restricted.

Example

Example 1: Liver fibrosis model mouse tree

Dimethylnitrosamine (DMN) was used as a liver fibrosis-inducing agent. DMN is known to affect the liver, causing liver dysfunction and cirrhosis, and is frequently used in rat and mouse experiments (Suzuki et al., Gene Ther 10, 765-7 73 (2003 itamura et al., Lab Invest 82, 571-583 (2002); Hata et al., J Histochem Cytochem 50, 1461-1468 (2002)). The liver recovers in a relatively short time even if inflammation with high regeneration ability occurs. However, fiber formation is induced by repeated cell death and regeneration due to inflammation. Therefore, we tried to construct a liver fibrosis model by repeatedly administering DMN every other day and causing chronic hepatitis-like symptoms. For the construction of liver fibrosis, BALB / c mice (female, 7 weeks old) were used, DMN (10 mg / kg) was administered twice every other day, and this administration condition was continued for 1 to 5 weeks (group). 1: This administration condition lasts for 1 week; Group 2: This administration condition lasts for 2 weeks; Group 3: This administration condition lasts for 3 weeks; Group 4: This administration condition lasts for 4 weeks; 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.

(1) Weight

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.

(2) GOT 'GPT value

Serum transaminase, Glutamic acid-Oxaloacetic Transaminase (GOT) and glutamic acid-pyruvate transaminase (GPT) in serum collected from each group of blood Therefore, we analyzed the tissue damage. As a result, GOT 'GPT increased in the DMN administration group in each group. In particular, the value was high in mice administered DMN for 3 weeks, and it was confirmed that there was a disorder in the monthly magazine.

(3) Accumulated amount of liver collagen 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.

Based on the above, it was confirmed that DMN administration for 3 weeks (group 3 administration conditions) was excellent in inducing liver fibrosis.

Example 2: Keying of baculovirus

(1) Baculovirus propagation

As a baculovirus, AcNPV (Autographa californica NPV), a nuclear polyhedrosis virus (NPV), was grown in a cultured cell system. Insect-derived Sf-9 cells were used as cultured cells. Sf-9 cells were seeded in a culture dish (φ 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.

(2) Recovery and concentration of baculovirus

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.

(3) Baculovirus titer measurement

The titer of baculovirus was determined by plaque assay. Sf-9 cells were seeded in a culture dish (φ 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-900Π 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. After adsorptive infection, the virus solution was removed, the agar medium was overlaid, and allowed to stand at 27 ° C for 72 hours to form plaques. The number of plaques formed was measured, and the virus titer (pfu / ml) was calculated by the following formula (I). Virus titer = [{(dilution rate) X (number of plaques)} / (number of dilution rate)] X 10 (I) Example 3: Treatment of liver fibrosis by baculovirus administration

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. To examine the effect of baculovirus on liver fibrosis, 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. Two days after baculovirus administration, dissection was performed, and blood and liver of the mice were collected and compared with DMN-induced liver fibrosis mice before administration. 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.

(1) Weight

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.

(2) GOT'GPT value

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.

(3) Accumulated amount of liver collagen

To examine how the amount of accumulated 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. However, the curovirus administration did not show a significant difference between the concentrations, but the value was significantly lower than that of physiological saline.

(4) Masson trichrome staining

In order to confirm the ability of collagen fiber formation to be improved by baculovirus administration, we performed Masson's trichrome staining and pathological analysis. Masson's trichrome stain 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.

(5) Hematoxylin and eosin staining

The cell state of the liver at this time was observed by HE staining. 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).

From the above, it was shown that baculovirus improved liver fibrosis.

Example 4: Biological response of mice by baculovirus administration

In 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 (10 ⁷ pfu / 200 μ 1 / animal), LPS (75)

2 hours, 12 hours, 24 hours, and 48 hours, and the abdominal lymph nodes, intraperitoneal cells, spleen and liver were collected. Noctomy virus and LPS were administered once or twice. Total RNA and DNA were extracted from the collected peritoneal lymph nodes, intraperitoneal cells, spleen and liver. In Example 3, since baculovirus was administered twice to examine the therapeutic effect, the liver recovered total RNA from the sample after twice baculovirus administration. To confirm baculovirus infection, vp39 gene expression was analyzed using PCR, and IFNs gene expression caused by baculovirus infection was analyzed using RT-PCR V. Figure 5 shows the actual dosing schedule.

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. Therefore, 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. In addition, 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. Gene expression of IFN-β was confirmed in the intestinal lymph node, intraperitoneal cells and spleen, and gene expression of IFN-γ was confirmed in intraperitoneal cells and spleen. The expression of the IFN-α 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.

Next, IFNs, HGF, and TGF-β 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. In addition, HGF, a liver regeneration factor, was strongly expressed after 2 hours. TGF-β gene expression decreased after 12 hours. This suggests that the HGF protein strongly induced by baculovirus administration suppresses TGF-β 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!

Based on the above, administration of baculovirus induces IFNs, which are liver fibrosis inhibitors, and HGF, a liver regeneration factor, and liver fibrosis is improved by suppressing the action of TGF-β, a liver fibrosis promoting factor. It was suggested that Therefore, it has been clarified that baculovirus can be effective in suppressing liver fibrosis and improving liver damage.

Industrial applicability

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.

This application is based on Japanese Patent Application No. 2006-275715 filed in Japan (filing date: October 6, 2006), the contents of which are incorporated in full herein.

Claims

The scope of the claims

[I] A preventive / therapeutic agent for liver damage, including baculovirus.

[2] The agent according to claim 1, wherein the baculovirus is a baculovirus vector.

[3] The agent according to claim 1 or 2, wherein the liver disorder is hepatitis, cirrhosis or liver cancer.

[4] An inhibitor of liver fibrosis, including baculovirus.

[5] Baculovirus inserted in a manner that allows expression of nucleic acids encoding factors that are effective in the prevention and treatment of liver disorders.

[6] Prevention of liver damage 'Prevention of the disease, which includes a baculovirus inserted in a manner capable of expressing a nucleic acid encoding a therapeutically effective factor and is applied to a subject suffering from liver damage · Therapeutic drugs.

[7] Concomitant medications, including baculovirus and preventive and therapeutic agents for liver damage.

[8] A kit containing a baculovirus and a prophylactic / therapeutic agent for liver damage.

[9] Use of baculovirus to produce a 'prevention of liver disorder' treatment.

[10] The use according to claim 9, wherein the baculovirus is a baculovirus vector.

[I I] Use according to claim 9 or 10, wherein the liver disorder is hepatitis, cirrhosis or liver cancer.

[12] Use of baculovirus to produce an inhibitor of liver fibrosis.

[13] In order to produce a nucleic acid that encodes an effective factor for the prevention and treatment of hepatic disorder to produce a 'preventive hepatic disorder' therapeutic agent applied to subjects suffering from hepatic disorder. The use of baculovirus.

[14] A method for preventing and treating liver damage in a subject, comprising administering an effective amount of baculovirus to the subject.

[15] The method according to claim 14, wherein the baculovirus is a baculovirus vector.

16. The method according to claim 14 or 15, wherein the liver disorder is hepatitis, cirrhosis or liver cancer.

[17] A method for suppressing liver fibrosis in a subject, comprising administering an effective amount of baculovirus to the subject.

[18] A subject suffering from liver damage should be administered an effective amount of baculovirus that has been inserted in a manner that allows expression of a nucleic acid encoding a factor effective in the prevention and treatment of liver damage! A method for preventing and treating liver damage in the subject.