KR101580313B1 - Composition for regenerating liver damage by infection of HBV comprising DNA Methyltransferase inhibitor - Google Patents

Abstract

The present invention relates to a composition for restoring liver damage caused by infection with HBV comprising DNA methyltransferase inhibitor as an active ingredient.

Description

Technical Field [0001] The present invention relates to a composition for recovering liver damage caused by infection with HBV comprising DNA methyltransferase inhibitor as an active ingredient.

The present invention relates to a composition for restoring liver damage caused by infection with HBV comprising DNA methyltransferase inhibitor as an active ingredient.

Infection with hepatitis B virus (HBV) causes many liver diseases including acute or chronic inflammation, cirrhosis and liver cancer. The chronic infection of HBV, which is estimated to infect approximately 400 million people worldwide, causes chronic inflammation of the liver causing liver damage (Feitelson, MA (1999).) Hepatitis B virus in hepatocarcinogenesis. J. Cell Physiol. 188-202). Long - term liver injury activates liver regeneration signals that supplement the reduction of hepatocytes. Hepatitis B virus in hepatocarcinogenesis. J. Cell Physiol. 181-208 (1981). Hepatitis B virus in hepatocarcinogenesis. J. Cell Physiol. 181-202 ). Thus, balanced liver regeneration is essential for liver homeostasis. In this regard, interference with liver regeneration by viral infection may be a major cause of multiple viral mediated liver disease.

Liver regeneration consists of very complex steps. Several growth factors (eg, hepatocyte growth factor (HGF), IL-6, TNF-alpha, and TGF-beta) are involved in regulating liver regeneration (Michalopoulos, GK, and DeFrances, MC (27), 60-66; Fausto, N., Campbell, JS, and Riehle, KJ (2006) Liver regeneration. Hepatology 43, S45-S53). Among them, HGF is known to have an action essential for liver regeneration. HGF is synthesized and secreted by an inactive single chain molecule (pro-HGF) and the hydrolytic cleavage of the protein of HGF results in the formation of two active chains, which have great affinity for c-met receptors (Naldini, L., Tamagnone, L., Vigna, E., Sachs, M., Hartmann, G., Birchmeier, W., Daikuhara, Y., Tsubouchi, H., Blasi, F., and Comoglio, PM (1992). , 4825-4833).

Korean Patent Publication No. 019910015292 discloses a pharmaceutical composition for accelerating liver tissue regeneration containing an effective amount of a macrolide compound or a salt thereof.

The present invention has been made in view of the above needs, and an object of the present invention is to provide a composition for regenerating liver injury caused by infection with HBV.

In order to accomplish the above object, the present invention provides a composition for repairing liver damage caused by infection with HBV comprising DNA methyltransferase inhibitor as an active ingredient.

In one embodiment of the present invention, the DNA methyltransferase is preferably composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The present invention also provides a composition for restoring hepatic injury caused by infection with HBV comprising 5-aza-2'-deoxycytidine as an active ingredient.

The present invention also provides a pharmaceutical composition for restoring liver damage caused by infection with HBV comprising 5-aza-2'-deoxycytidine as an active ingredient.

The present invention also provides a functional food composition for restoring liver damage caused by infection with HBV comprising 5-aza-2'-deoxycytidine as an active ingredient.

In one embodiment of the invention, a therapeutically effective amount of a compound of the invention is used in admixture with a pharmaceutically acceptable carrier. Such compositions are generally administered by parenteral injection or infusion. Subcutaneous, intravenous, or intramuscular injection routes are selected depending on the therapeutic effect achieved.

When administered systemically, the composition for use in the present invention is in the form of a parenterally acceptable aqueous solution without a heat source. Pharmaceutically acceptable sterile protein solutions, including pH, isotonicity, stability, carrier protein and the like, are within the skill of the art.

Also included within the present invention are pharmaceutical compositions comprising an effective amount of the compounds of the present invention together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers useful in therapy. As used herein, the term " therapeutically effective amount " refers to an amount that represents a regenerative effect for a given condition and dosage regimen. Such compositions are liquids, gels, ointments, or lyophilized or otherwise dried formulations and may contain various buffering ingredients (e.g., Tris-HCl, acetate, phosphate), diluents with pH and ionic strength, (E.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycol), antioxidants (such as ascorbic acid, sodium metabisulfite) (Eg, thimerosal, benzyl alcohol, parabens), bulky substances or rigid modifiers (eg, lactose, mannitol), covalent attachments to proteins of polymers such as polyethylene glycols, Granules of polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels and the like, Microspheres, microemulsions, micelles, monolayer or multi-layer vesicles, in vivo degradable injectable microcapsules or microspheres, or protein matrices, erythrocyte ghosts, Or other known method of preparation or packaging of pharmaceuticals.

Other embodiments of the compositions of the present invention incorporate a granular form of a protective coating, protease inhibitor, or permeation enhancer for various routes of administration including parenteral, pulmonary, intranasal, topical (skin or mucosal) and oral.

The compositions of the invention are packaged in sterile vials or ampoules in unit dosage form.

The compositions of the present invention can be incorporated into microswitches in an isotropically clear liquid dispersion of two non-miscible liquids, such as oil and water, which is generally thermodynamically stable and stabilized by the interfacial membrane of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). Surfactants (emulsifiers), co-surfactants (co-emulsifiers), oil phase, and water phase are essential for microsurgical manufacturing. Suitable surfactants are any surfactants useful in emulsion preparation, such as emulsifiers commonly used in the manufacture of creams. The co-surfactant (or "co-emulsifier") is generally selected from the group of polyglycerol derivatives, glycerol derivatives and fatty alcohols. Preferred emulsifier / co-emulsifier combinations generally include glyceryl monostearate and polyoxyethylene stearate; Polyethylene glycol and ethylene glycol palmitostearate; Caprylic and capric triglycerides, and oleyl macrogol glycerides, but not necessarily. The aqueous phase includes not only water but also buffers, glucose, propylene glycol, polyethylene glycols, preferably low molecular weight polyethylene glycols (e.g., PEG 300 and PEG 400), and / or glycerol and the like, For example, fatty acid esters, modified vegetable oils, silicone oils, mono-, di- and triglycerides, mono- and diesters of PEG (e.g. oleic macrogolglycerides) and the like.

The pharmaceutical preparations of the present invention can be conventionally provided in unit dosage forms, which can be prepared according to techniques known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier or excipient. Generally, formulations can be made by bringing the active ingredients into association with a liquid carrier or a finely divided carrier, or both, and then shaping the product as needed.

The compositions of the present invention may be prepared in many possible forms including but not limited to tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enema. The compositions of the present invention may be formulated as suspensions in liquid, non-liquid or mixed media. The aqueous suspension may further comprise a substance which increases the viscosity of the suspension comprising carboxymethylcellulose, sorbitol and / or dextran. The suspension may contain a stabilizer.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, xerox, liposome-containing preparations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

Compositions and formulations for oral administration include suspensions or solutions in powders or granules, microparticles, nanoparticles, water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini tablets. Thickeners, flavorings, diluents, antiseptics, dispersion aids or binders may be preferred. Preferred oral formulations are those that are administered with the oligonucleotides of the invention and one or more penetration enhancers, surfactants and chelators. Preferred surfactants include fatty acids and / or esters or salts thereof, bile acids and / or salts thereof. Preferred bile acids / salts and fatty acids and their uses are described in U.S. Pat. Is further described in patent 6,287, 860, which is incorporated by reference. Penetration enhancers, for example fatty acid / salt and bile acid / salt complexes, are also preferred. A particularly preferred combination is lauric acid, capryic acid and the sodium salt of UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. When the composition of the present invention comprises an oligonucleotide, it can be orally transported into a granular form containing sprayed dried granules, or can be compounded to form fine particles or nanoparticles. Oligonucleotide < / RTI > Which is further described in patent 6,287,860.

Compositions and formulations for intravenous, intramuscular or intraventricular administration include sterile aqueous solutions, including but not limited to buffers, diluents and penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients, .

The appropriate amount will depend on the course of the treatment which lasts from a few days to several months, or the severity of the disease condition to be treated, as well as the course of treatment up to the day that the treatment effect or reduction in the disease state is obtained. The optimal drug injection schedule can be calculated from the drug accumulation measurements on the patient ' s body. Those skilled in the art can readily determine the optimum dose, method and repetition rate. Optimal doses can vary with relative efficacy and are generally predicted based on the EC50s found to be effective in in vitro and in vivo animal models. Generally, the dosage will be from 0.01 μg to 10 g per kg of body weight, administered more than once per day, and the skilled artisan will readily determine the repetition rate for drug infusion based on the measured residence time and the concentration of the drug in body fluids or tissues It will be possible. After successful treatment, it is desirable to have the patient undergo maintenance therapy to prevent recurrence of the disease state.

The present invention also provides that the pharmaceutical composition of the present invention is packaged in an enclosed sealed container such as an ampoule or sachet indicating the amount of formulation. In one embodiment, the pharmaceutical compositions of the present invention are provided as anhydrous sterile lyophilized powder or water-free concentrate in an enclosed sealed container and reconstituted to an appropriate concentration for administration to the subject (e. G., Water or saline ). ≪ / RTI > Preferably, the pharmaceutical composition of the present invention comprises at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, mg, at least 50 mg, at least 75 mg, or at least 100 mg. The lyophilized pharmaceutical composition of the present invention may be stored in its original container at 2 캜 to 8 캜 and the pharmaceutical composition of the present invention may be reconstituted for 1 week, preferably within 5 days, within 72 hours, for 48 hours Within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, the pharmaceutical composition of the present invention is supplied in a liquid form in an enclosed sealed container representing the quality and concentration of the formulation. Preferably, the liquid form of the administered composition is at least 0.25 mg / ml, more preferably at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml or at least 100 mg / ml. The liquid form should be stored in its original container at 2 캜 to 8 캜.

Hereinafter, the present invention will be described.

The present inventors have investigated the molecular mechanisms for inhibition of liver repair using liver cell lines and mouse models. The present inventors have established a mouse model of acute HBV infection by hydrodynamic injection of viral DNA. Liver restoration after partial hepatectomy was significantly inhibited in the mouse model of HBV infection. Mechanism studies showed that the expression of urokinase-type plasminogen activator (uPA), which regulates the activation of hepatocyte growth factor (HGF), was significantly reduced in liver tissues of HBV or HBx-expressing mice. Downregulation of uPA was confirmed using HBx and HBV genomic transiently or stably transfected liver cell lines. In addition, the present inventors have shown that HBx inhibits uPA expression through transgenic control of the uPA promoter in mouse liver and human liver cell lines. Expression of HBx strongly induced hypermethylation of the uPA promoter by recruiting DNMT3A2.

DNMT3A2 The HBx - Induced uPA Down to adjust  Involved

To demonstrate the relevance of DNMT activity to HBx-induced inhibition of uPA, we treated Huh7-HBx cells with the DNMT inhibitor 5-aza-2'-deoxycytidine (5-AzaC). Luciferase assays showed that uPA promoter activity was restored by 5-AzaC treatment (Fig. 1A). Semiquantitative RT-PCR analysis also showed that 5-AzaC treatment significantly restored uPA expression (Fig. 1b). These results indicate that inhibition of uPA by HBx is regulated by aerobic control of the uPA promoter.

HBV From In Vivo uPA of Hygienic  Regulation inhibits liver repair

To observe whether inhibition of hepatocyte proliferation by HBV and HBx can be observed in in vivo, the present inventors established a mouse model of acute HBV infection by hydrodynamic injection of viral DNA. pHBV1.2 (wt) or pHBV1.2 (HBx-) plasmids were hydraulically injected into the tail vein of mice.

To determine whether liver expression of HBx affects liver repair, we investigated the state of the recovered liver after partial hepatectomy of HBV model mice. Importantly, the recovered liver after 8 days of partial hepatectomy showed a significant reduction only in the mouse model of wt HBV infection, whereas the HBx-null HBV-infected mice showed normal liver resection (FIG. 2A). Similar results were also observed from a mouse model of HBx expression (Figure 2B). These results indicate that HBx expression inhibits liver repair after partial hepatectomy.

To determine whether HBV or HBx affects liver function, we analyzed the epidemiology of serum AST, ALT, total protein and albumin after partial hepatectomy. There were no significant differences among the groups at all time points we tested (Figure 2C).

To investigate the epidemiological changes of uPA by HBx in the mouse liver, we determined the activity and expression of uPA at several time points after partial hepatectomy. As shown in Figures 3A and 3B, wt HBV retarded the activity and expression of uPA and was significantly inhibited.

In order to assess whether other pro-restorative factors such as IL-6 and TNF-alpha are involved in this model system, we performed a kinetic analysis of IL-6 and TNF-alpha after partial hepatectomy. These cytokines increased at the initial site. However, there is no significant difference in the experimental group (Fig. 3C, D), suggesting that the uPA pathway is primarily involved in this system.

To confirm this, we investigated the effect of HBx on liver recovery in uPA knockdown mice. The present inventors have found that HBx did not show any additional effect in uPA knockdown mice (Fig. 3E, F). Taken together, these results indicate that the uPA pathway is primarily involved in HBV-mediated inhibition of liver repair.

DNA methyltransferase  ( DNMT ) Inhibitor treatment recovers liver repair from in vivo

The activity of DNMT is essential for HBV mediated inhibition of liver repair. Therefore, the present inventors investigated whether inhibition of DNMT restored liver repair. The present inventors inhibited DNMT activity by injection of 5'AzaC in mouse tail vein as shown in FIG. 4A. Down-regulated uPA expression by HBx in mouse liver was increased by 5'AzaC treatment (Fig. 4B). Liver restoration was almost completely restored by the treatment of DNMT inhibitors (Figure 4C).

The resolution of the molecular basis of inhibition of liver regeneration by HBV infection through the present invention not only extends the understanding of the hospital physiology of viral mediated liver disease, but also ultimately provides new therapeutic options for patients with liver disease will be.

Figure 1 shows that DNMT3A2 is involved in HBx-induced uPA down regulation and in combination with HBx to recruit on the uPA promoter
(a) Relative luciferase activity of uPA-luc in Huh7 cells transfected with pHBV1.2 (wt) or pHBV1.2 (HBx-) after 5-aza-2'-deoxycytidine (5AzaC) treatment with the DNA methyltransferase inhibitor Say. Data represent the mean ± SD of three independent experiments. (b) Expression of uPA mRNA after 5AzaC treatment in Huh7-HBx stable cells.
Figure 2 shows that HBx inhibits liver repair in a mouse model of HBV infection. (A, B) liver repair was inhibited by HBx in an HBV mouse model.
Macroscopic evaluation of recovered liver of HBV or HBx mouse model after 8 days of partial hepatectomy. Box drawings of liver weight / weight after 8 days of partial hepatectomy. (C) Liver function test after partial hepatectomy
Figure 3 shows the kinetic analysis of uPA, IL-6 and TNF-alpha after partial hepatectomy. (A) Kinetic analysis of uPA foot movement in liver tissue after partial hepatectomy with Western blot. (B) Kinetic analysis of uPA activity in liver tissue after partial hepatectomy by casein-plasminogen biomarkers at the time points listed. (C) Kinetic analysis of serum IL-6 (C) and TNF-alpha (D) after partial hepatectomy at the time points indicated. (E) Knockout of uPA expression in mouse liver by hydrodynamic injection of siuPA. (F) HBx effect of liver repair in uPA knockdown mice Partial liver resection was performed 4 days after the simultaneous hydrodynamic injection of HBV plasmid and siuPA. At least three mice were used in the analysis of each group.
Figure 4 shows that inhibition of DNMT activity restores liver repair in vivo. (A) Experimental scheme for 5'AzaC treatment (5 mg / kg injection before partial liver resection, 1 mg / kg injection after partial liver resection). (B) Analysis of uPA expression in liver tissue after 5'AzaC injection. (C) Box chart of relative liver resection after 8 days of partial liver resection. At least three mice were used in the analysis of each group. HI, hydraulic injection; PH, partial hepatectomy.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are provided to illustrate the present invention and the scope of the present invention is not construed as being limited by the following examples.

Example  1: In the mouse Plasmid DNA of Hydrodynamic  injection

6-7 week old male BALB / c mice were used for the hydraulic injection of plasmid DNA. Mice were hydrodynamically injected into 25 μg of plasmid DNA (pHBV1.2 (wt), pHBV1.2 (HBx-), pCMV-HBx-HA and pCMV-HA) in tail vein to a volume of 10% . A total volume of DNA was delivered to the vein at high pressure within 4-6 seconds (hydro-vibrotransfection).

For knockout of in vivo uPA, the mice were hydraulically injected with 25 ug of siuPA (5'-CUCAUCUUGCACGAAUACU-3 '). To restore uPA expression in the liver of HBV-expressing mice, 25ug of mouse uPA vector was hydraulically injected with HBV1.2 (wt). Two days later, the same amount of uPA vector was injected additionally. All animal experiments were approved by the Animal Ethics Committee at Konkuk University.

Example  2: Casein Zymography

The activity of uPA was analyzed by direct caseinization. Briefly, samples were mixed with a sample buffer without reducing agent such as beta-mercaptoethanol and then mixed with casein (1 mg / ml; Sigma, St. Louis, MO) and plasminogen (13 μg / ml; American Diagnostica, Stamford, CT) On a 10% polyacrylamide gel. The casein degradation band detected at 52 kDa is specific for uPA since it corresponds to standard uPA (Chemicon, Temecula, CA).

Example  3: Western Blot  analysis

Cells were disrupted in disruption buffer (25 mM Tris-HCl (pH 7.5), 1% NP40, and protease inhibitor cocktail) and centrifuged. The supernatant was collected and heated to 95 < 0 > C for 3 minutes. Each of the tissues was treated with RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1% Triton X-100, 0.5 mM DTT, 100 μg / ml phenylmethylsulfonyl fluoride, 0.3 μM Aprotinin, Leupeptin, 2 μM Pepstatin, 1.3 mM EDTA-Na ₂ , 2 mM Na ₃ VO ₄ , and 10 μg / ml E64). The tissue lysate was centrifuged at 13000 g for 30 minutes and the supernatant was collected. After heating, the protein was quantitated by Bradford protein assay Reagent (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin (10 mg / mL) as a standard. 50 μg of protein was separated by 12% SDS-PAGE gel and Western blot analysis was performed. The primary antibodies used were anti-uPA (M-20, Santa Cruz Biotechnology, CA, USA), anti-HGF (Santa Cruz Biotechnology) and anti-beta-actin (Sigma Aldrich). As secondary antibody, a suitable peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) was used. Chemiluminant detection was performed using an ECL Detection Reagent (GE Healthcare, Buckinghamshire, UK) and visualized with an X-ray film or Bio-Imaging analyzer (LAS-3000, Fuji, Tokyo, Japan).

Example  4: Liver Function Test

The mice were sacrificed after partial hepatectomy at the indicated time points (0, 2, 6, 12, 24 and 48 hours). Blood samples were collected at each time point and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), whole protein and albumin were analyzed by the Cobas 8000 C702 system using the Roche Diagnostic Kit. At least three mice were used in the analysis of each group.

Example  5: IL -6 and TNF - Measurement of Alpha

The mice were sacrificed after partial hepatectomy at the indicated time points (0, 2, 6, 12, 24 and 48 hours). Blood samples were collected at each time point and serum levels of IL-6 and TNF-alpha were analyzed using mouse IL-6 and TNF-alpha ELISA kits (R & D Systems, Minneapolis, Minnesota) according to the manufacturer's instructions. At least three mice were used in the analysis of each group.

Example  6: 5- AzadC  Processing and bisulfite DNA  Sequencing

Freshly prepared 5-AzadC (Sigma-Aldrich Co, St. Louis, Mo.) at a final concentration of 10 mmol / L was added to the culture medium and replaced every 24 hours for 3 days for DNA methylation inhibition studies.

Sodium bisulfite strain was performed on genomic DNA as follows. In summary, 2 μg of genomic DNA was denatured with 2 M NaOH at 37 ° C for 15 minutes and incubated with 3 M sodium bisulfite (pH 5.0) for 16 hours at 50 ° C. After purification, the modified DNA was amplified by PCR. The PCR product was gel-extracted and subcloned into pGEM-T easy vector (Promega). Five to ten clones per sample were selected for DNA sequencing.

All data were obtained at least three independent experiments and the values were expressed as mean ± SD. Differences between groups were tested for statistical significance using analysis of variance or Student's t- test. Statistical significance was set at p <0.05.

<110> Konkuk University Industrial Cooperation Corp. <120> Composition for regenerating liver damage by infection of HBV          comprising DNA Methyltransferase inhibitor <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 912 <212> PRT <213> Homo sapiens <400> 1 Met Pro Ala Met Pro Ser Ser Gly Pro Gly Asp Thr Ser Ser Ser Ala   1 5 10 15 Ala Glu Arg Glu Glu Asp Arg Lys Asp Gly Glu Glu Gln Glu Glu Pro              20 25 30 Arg Gly Lys Glu Glu Arg Gln Glu Pro Ser Thr Thr Ala Arg Lys Val          35 40 45 Gly Arg Pro Gly Arg Lys Arg Lys His Pro Pro Val Glu Ser Gly Asp      50 55 60 Thr Pro Lys Asp Pro Ala Val Ile Ser Lys Ser Pro Ser Met Ala Gln  65 70 75 80 Asp Ser Gly Ala Ser Glu Leu Leu Pro Asn Gly Asp Leu Glu Lys Arg                  85 90 95 Ser Glu Pro Gln Pro Glu Glu Gly Ser Pro Ala Gly Gly Gln Lys Gly             100 105 110 Gly Ala Pro Ala Glu Gly Glu Gly Ala Ala Glu Thr Leu Pro Glu Ala         115 120 125 Ser Arg Ala Val Glu Asn Gly Cys Cys Thr Pro Lys Glu Gly Arg Gly     130 135 140 Ala Pro Ala Glu Ala Gly Lys Glu Gln Lys Glu Thr Asn Ile Glu Ser 145 150 155 160 Met Lys Met Glu Gly Ser Arg Gly Arg Leu Arg Gly Gly Leu Gly Trp                 165 170 175 Glu Ser Ser Leu Arg Gln Arg Pro Met Pro Arg Leu Thr Phe Gln Ala             180 185 190 Gly Asp Pro Tyr Tyr Ile Ser Lys Arg Lys Arg Asp Glu Trp Leu Ala         195 200 205 Arg Trp Lys Arg Glu Ala Glu Lys Lys Ala Lys Val Ile Ala Gly Met     210 215 220 Asn Ala Val Glu Glu Asn Gln Gly Pro Gly Glu Ser Gln Lys Val Glu 225 230 235 240 Glu Ala Ser Pro Ala Val Gln Gln Pro Thr Asp Pro Ala Ser Pro                 245 250 255 Thr Val Ala Thr Thr Pro Glu Pro Val Gly Ser Asp Ala Gly Asp Lys             260 265 270 Asn Ala Thr Lys Ala Gly Asp Asp Glu Pro Glu Tyr Glu Asp Gly Arg         275 280 285 Gly Phe Gly Ile Gly Glu Leu Val Trp Gly Lys Leu Arg Gly Phe Ser     290 295 300 Trp Trp Pro Gly Arg Ile Val Ser Trp Trp Met Thr Gly Arg Ser Arg 305 310 315 320 Ala Ala Glu Gly Thr Arg Trp Val Met Trp Phe Gly Asp Gly Lys Phe                 325 330 335 Ser Val Val Cys Val Glu Lys Leu Met Pro Leu Ser Ser Phe Cys Ser             340 345 350 Ala Phe His Gln Ala Thr Tyr Asn Lys Gln Pro Met Tyr Arg Lys Ala         355 360 365 Ile Tyr Glu Val Leu Gln Val Ala Ser Ser Ala Gly Lys Leu Phe     370 375 380 Pro Val Cys His Asp Ser Asp Glu Ser Asp Thr Ala Lys Ala Val Glu 385 390 395 400 Val Gln Asn Lys Pro Met Ile Glu Trp Ala Leu Gly Gly Phe Gln Pro                 405 410 415 Ser Gly Pro Lys Gly Leu Glu Pro Pro Glu Glu Glu Lys Asn Pro Tyr             420 425 430 Lys Glu Val Tyr Thr Asp Met Trp Val Glu Pro Glu Ala Ala Ala Tyr         435 440 445 Ala Pro Pro Pro Ala Lys Lys Pro Arg Lys Ser Thr Ala Glu Lys     450 455 460 Pro Lys Val Lys Glu Ile Ile Asp Glu Arg Thr Arg Glu Arg Leu Val 465 470 475 480 Tyr Glu Val Arg Gln Lys Cys Arg Asn Ile Glu Asp Ile Cys Ile Ser                 485 490 495 Cys Gly Ser Leu Asn Val Thr Leu Glu His Pro Leu Phe Val Gly Gly             500 505 510 Met Cys Gln Asn Cys Lys Asn Cys Phe Leu Glu Cys Ala Tyr Gln Tyr         515 520 525 Asp Asp Asp Gly Tyr Gln Ser Tyr Cys Thr Ile Cys Cys Gly Gly Arg     530 535 540 Glu Val Leu Met Cys Gly Asn Asn Asn Cys Cys Arg Cys Phe Cys Val 545 550 555 560 Glu Cys Val Asp Leu Leu Val Gly Pro Gly Ala Ala Gln Ala Ala Ile                 565 570 575 Lys Glu Asp Pro Trp Asn Cys Tyr Met Cys Gly His Lys Gly Thr Tyr             580 585 590 Gly Leu Leu Arg Arg Arg Glu Asp Trp Pro Ser Arg Leu Gln Met Phe         595 600 605 Phe Ala Asn Asn His Asp Gln Glu Phe Asp Pro Pro Lys Val Tyr Pro     610 615 620 Pro Val Pro Ala Glu Lys Arg Lys Pro Ile Arg Val Leu Ser Leu Phe 625 630 635 640 Asp Gly Ile Ala Thr Gly Leu Leu Val Leu Lys Asp Leu Gly Ile Gln                 645 650 655 Val Asp Arg Tyr Ile Ala Ser Glu Val Cys Glu Asp Ser Ile Thr Val             660 665 670 Gly Met Val Arg His Gln Gly Lys Ile Met Tyr Val Gly Asp Val Arg         675 680 685 Ser Val Thr Gln Lys His Ile Gln Glu Trp Gly Pro Phe Asp Leu Val     690 695 700 Ile Gly Gly Ser Pro Cys Asn Asp Leu Ser Ile Val Asn Pro Ala Arg 705 710 715 720 Lys Gly Leu Tyr Glu Gly Thr Gly Arg Leu Phe Phe Glu Phe Tyr Arg                 725 730 735 Leu Leu His Asp Ala Arg Pro Lys Glu Gly Asp Asp Arg Pro Phe Phe             740 745 750 Trp Leu Phe Glu Asn Val Val Ala Met Gly Val Ser Asp Lys Arg Asp         755 760 765 Ile Ser Arg Phe Leu Glu Ser Asn Pro Val Met Ile Asp Ala Lys Glu     770 775 780 Val Ser Ala Ala His Arg Ala Arg Tyr Phe Trp Gly Asn Leu Pro Gly 785 790 795 800 Met Asn Arg Pro Leu Ala Ser Thr Val Asn Asp Lys Leu Glu Leu Gln                 805 810 815 Glu Cys Leu Glu His Gly Arg Ile Ala Lys Phe Ser Lys Val Arg Thr             820 825 830 Ile Thr Thr Arg Ser Asn Ser Ile Lys Gln Gly Lys Asp Gln His Phe         835 840 845 Pro Val Phe Met Asn Glu Lys Glu Asp Ile Leu Trp Cys Thr Glu Met     850 855 860 Glu Arg Val Phe Gly Phe Pro Val His Tyr Thr Asp Val Ser Asn Met 865 870 875 880 Ser Arg Leu Ala Arg Gln Arg Leu Leu Gly Arg Ser Trp Ser Val Pro                 885 890 895 Val Ile Arg His Leu Phe Ala Pro Leu Lys Glu Tyr Phe Ala Cys Val             900 905 910

Claims (5)

delete delete delete 5-aza-2'-deoxycytidine as an active ingredient and injected with 5 mg / kg of 5-aza-2'-deoxycytidine before partial hepatectomy and 1 mg / kg after partial hepatectomy Wherein the hepatitis B virus (HBV) infection is a hepatitis B virus infection.
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