KR20180105084A - Novel Use of Rigosertib for treating Hepatitis C Virus Disease - Google Patents

Abstract

The present invention relates to a novel use of rigosertib and, more specifically, relates to a use of rigosertib for preventing or treating a liver disease caused by hepatitis C virus and for resisting hepatitis C virus, capable of effectively and specifically inhibiting HCV infection without making issues about tolerance. According to the present invention, rigosertib is able to be used as a specific and effective medicine for HCV infection since the rigosertib inhibits a bond between micro RNA-122 and ELAVL1/HuR. Moreover, unlike conventional anti-HCV medicines, since HCV protein is not targeted and the activity of interstitial cell functioning molecules is inhibited, existing tolerance issues are able to be solved, and thus, the rigosertib is able to be solely used or used in parallel with a conventional medicine as a good HCV treatment solution which does not cause tolerance issues.

Description

{Novel Use of Rigosertib for Treating Hepatitis C Virus Disease}

The present invention relates to novel uses of lysoser tips, and more particularly to the use of Lysozyme tip for the prevention or treatment of hepatitis C caused by hepatitis C virus and the use of antiviral against hepatitis C virus.

Rigosertib (Onconova Therapeutics, Inc.) is a synthetic benzylstyrylsulfone substance previously known as an anticancer drug. It is effective in the treatment of proliferative diseases (US7598232), myelodysplastic syndrome and acute myelogenous leukemia treatment (US8664272) Is known. However, there has been no report of use related to liver disease, and furthermore, hepatitis C virus.

Drug Repositioning is a concept to develop new indications for drugs currently in use and is recognized as an alternative to dramatically improve the success of new drug development. New drug re-creation is likely to succeed in the development of new drugs because it can shorten the cost and duration of new drug development if the safety of new drug candidates is verified and validated. Re-creation of new medicines means: (i) the use, preparation and stability of the drug are established; (Ii) ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) data, ie, drug absorption rate, diffusion, metabolism, emissions and drug toxicity information are established; (Iii) the likelihood of failure due to safety issues is significantly low based on the results of clinical trials of the drug before the drug use change; (Iv) Post-marketing surveillance (PMS) has the advantage that it does not have to spend time securing safety data.

On the other hand, hepatitis C is mostly symptomless at the early stage of infection and it is known that when infected with an adult, chronic hepatitis is over 75%, unlike infection with hepatitis B virus. Chronic hepatitis C is a disease in which the immune system does not completely eliminate the hepatitis C virus and the infection lasts for more than 6 months, and chronic inflammation of the liver persists.

Hepatitis C virus (HCV) is an RNA virus classified as a hepacivirus genus of the Flaviviridae, which was discovered in the United States in 1989. It is a clinically important virus that causes hepatitis, cirrhosis and hepatocellular carcinoma. In developed countries like USA and Europe, HCV is the main cause of chronic hepatitis. According to the WHO, hepatitis C virus (HCV) is becoming a more serious problem, with approximately 200 million people infected with more than 3% of the world population and more than 3-4 million additional infections every year .

In particular, it is rare that the virus disappears naturally without treatment, and 5 ~ 20% progress to cirrhosis after a period of 20-25 years, and patients progressing to cirrhosis progress to liver cancer with an incidence of 1 ~ 5% per year . About 12% of patients with liver cirrhosis and 15% of patients with hepatocellular carcinoma are known to be caused by hepatitis C virus and are the main cause of chronic liver disease together with hepatitis B virus.

To date, widely used as a treatment for HCV is co-therapy with peginterferon and ribavirin, with a treatment rate of only 30-50%, which is also dependent on the genotype of HCV It is different. Recently, therapies targeting HCV non-structural proteins such as Sovaldi and Harvoni of Gilead Sciences have been reported, but they are expensive to apply as a treatment for most patients.

The development of antiviral agents is continuously required to overcome the emergence of resistant viruses, side effects of drugs, and high production costs. In the case of HCV, a system capable of producing an infectious virus has not been developed for a long time, and mutation rate is high, and frequent variants are emerging. Therefore, it is urgently required to develop a therapeutic drug widely acting as a new mechanism of action.

Accordingly, the present inventors have made efforts to develop a therapeutic or prophylactic agent for a liver disease caused by a novel HCV infection that can solve the problem of resistance to existing drugs while specifically and effectively acting on HCV infection, and as a result, it plays an indispensable role in replication of hepatitis C virus We found that ELAVL1 / HuR, an intracellular molecule that specifically binds to miRNA-122, is known to bind specifically and strongly to sirtip. It has been confirmed that inhibiting the binding of miRNA-122 and ELAVL1 / HuR important for translation / replication has a pharmaceutical use as an anti-HCV therapeutic agent as a specific and effective inhibitor against HCV infection, and the present invention has been completed.

The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .

Patent Document 1: US 7598232 B Patent Document 2: US 8664272 B

It is an object of the present invention to provide a pharmaceutical composition capable of preventing or treating liver disease caused by the novel hepatitis C virus.

Another object of the present invention is to provide an antiviral composition against hepatitis C virus.

It is still another object of the present invention to provide a health functional food composition capable of preventing or ameliorating liver disease caused by hepatitis C virus.

In order to achieve the above object, the present invention provides a new use of the lyogor tip.

Specifically, the present invention relates to a pharmaceutical composition for the treatment or prevention of hepatitis C caused by hepatitis C virus (HCV), which comprises, as an active ingredient, rigosertib or a pharmaceutically acceptable salt thereof .

The present invention also provides an antiviral composition for HCV comprising lysogliptin or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health functional food composition for improving or preventing liver disease caused by HCV, which contains Lysozyme tip or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a novel antiviral agent against HCV and a composition for preventing or treating liver disease caused by HCV that can specifically and effectively inhibit HCV infection without resistance problems.

The ligase tip according to the present invention can be a specific and effective therapeutic agent for HCV infection by inhibiting the binding of microRNA-122 and ELAVL1 / HuR. Furthermore, unlike the previously developed anti-HCV therapeutic agents, it inhibits the action of human hepatocyte action molecules without targeting the HCV protein, and thus can solve the resistance problem against the previously developed drugs, Combination therapy with known therapeutic agents is possible and is useful as a good treatment strategy for HCV that does not cause resistance.

1 shows miRNA-122 directly binding to ELAVL1 / HuR and results of ELAVL1 / HuR efficacy assays. 1A is the isomiR structures of miRNA-122 and 1B is an RNA electro mobility shift assay using GST-HuR ) 1C is the result of assaying the intracellular molecule ELAVL1 / HuR using an HCV subgenomic replicon capable of translation / replication assay, and 1D specifically knockout ELAVL1 / HuR in Huh7 cells using CRISPR / Cas9 system This is an image of the resulting Westingblatt.
FIG. 2A is a graph showing a screening result of a compound inhibiting the HCV translation / proliferation process using the structure of a subgenomic replicon of HCV capable of a reporter assay (HIRL-2Aneo-NSrep) and a Seleckchem cellular kinase inhibitor libray, and 2B is a graph showing a result of screening of recombinant GST- And biotin-rigosertb by in vitro pulldown assay, 2C and 2D represent the chemical structure of lysophospholipid and biotin-ligosertb, 2E is the interaction between intracellular molecules and HCV (ELAVL1 / HuR-microRNA-122-HCV translation / replication).
Figure 3 is a graph re-verifying the anti-HCV effect of Lysozyme tip through HIRL-2Aneo-NSrep_FL Huh-7 luciferase assay.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The definitions of the main terms used in the description of the present invention and the like are as follows.

The term "treatment" in the present invention means an approach for obtaining a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Which may be partial or total), detectable or undetected. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include not only the disorder to be prevented, but also the treatment required for the already existing field. "Palliating " a disease means that a range of disease states and / or undesirable clinical manifestations are reduced or the time course of the progress is slowed or lengthened, as compared to no treatment.

The term "prevention" in the present invention refers to a therapy that protects against the onset of a disease or disorder so that the clinical symptoms of the disease are not developed. Thus, "prophylactic" is intended to include administering a therapy to a subject (e.g., administering a therapeutic agent) before the manifestation of the disease is detectable in the subject (e.g., Virus) in the absence of the therapeutic agent. The subject may be an individual having one or more risk factors known to be associated with the development or onset of an individual, such as a disease or disorder, at risk of developing the disease or disorder. Thus, in certain embodiments, the term "preventing HCV infection" refers to administering an anti-HCV therapeutic agent to a subject that does not have a detectable HCV infection. It is understood that the subject for anti-HCV prophylaxis may be an individual at risk for HCV virus.

The term "therapeutically effective amount" or "effective amount ", as used herein, refers to an amount effective to elicit the desired biological or medical response, such as the amount of a compound sufficient to cause such treatment for a disease when administered to a subject for treating the disease Quot; The effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc. of the subject to be treated. An effective amount may include a positive range. As will be appreciated in the pertinent art, an effective amount may be at least one dose, i.e. a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. An effective amount can be considered in the context of administering one or more therapeutic agents and a single agent can be considered to be provided in an effective amount when desirable or beneficial results can be achieved or achieved with one or more other agents. The appropriate dose of any co-administered compound may be optionally reduced due to the combined action of the compound (e. G., Additional or synergistic effect).

Throughout this specification, unless the context requires otherwise, the words "comprises" and "comprises" include inclusive reference to a group of steps or steps suggested but not to the exclusion of any other step or group of steps I must understand.

Hereinafter, the present invention will be specifically described.

In one aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of liver disease caused by hepatitis C virus (HCV) comprising, as an active ingredient, rigosertib or a pharmaceutically acceptable salt thereof. ≪ / RTI >

Rigosertib is a substance known as an anticancer drug, specifically a substance having a structure represented by the following formula (1).

[Chemical Formula 1]

Hepatitis C virus consists of single-stranded single stranded (+) sense RNA of molecular biology, belongs to flaviviridae, uses an abnormal translation initiation process such as internal ribosomal entry site, The translation process occurs and is then cleaved by proteases encoded by intracellular proteases or viruses, respectively, to designate non-structural proteins important for translation, replication, and proliferation of structural proteins or viruses, respectively . Previous studies have shown that HCV replication and translation processes actively utilize intracellular molecules, and microRNA-122 (miRNA-122, one of the intracellular molecules, accounts for 70% of microRNAs in human hepatocytes ) Is well known to play an indispensable role in the replication process of hepatitis C virus.

In the examples of the present invention, it was confirmed that the linkage of miRNA-122 and ELAVL1 / HuR and the molecule ELAVL1 / HuR, which was found to be important for HCV translation and replication, Serte tip has been shown to inhibit translation / replication / proliferation of HCV by inhibiting the direct binding of miRNA-122, an intracellular molecule, to the ELAVL1 / HuR molecule, which is known to play an important role in the replication process of HCV translation.

In another embodiment of the present invention, it has been confirmed that ligoser tip strongly inhibits replicon proliferation similar to danoprevir that has already been developed in Roche and used in clinical use, and exhibits an inhibitory effect of 80% or more even at a low concentration (0.2 μM).

The liposome tip of the present invention inhibits the action of a host factor (a molecule of human hepatocyte) rather than a protein produced by HCV itself unlike the existing anti-HCV therapeutic agent (miRNA-122 and ELAVL1 / HuR) and can be used as a preventive or therapeutic agent for hepatitis C due to the novel concept of hepatitis C virus, which can solve the resistance problems against drugs that have been developed, since they do not target HCV protein.

The liver disease caused by the hepatitis C virus may be hepatitis C, hepatic fibrosis caused by hepatitis C virus, liver cirrhosis caused by hepatitis C virus, and liver cancer caused by hepatitis C virus.

At this time, the ligase tip may be characterized in that it is administered in combination with an HCV therapeutic agent, for example, sovaldi, danoprevir, or Daclatasvir.

It can also be administered in combination with an antiviral agent, for example, ribavirin.

In addition, Lysozyme tip inhibits the translation / replication / proliferation of hepatitis C virus and can be used as an antiviral agent against hepatitis C virus. Thus, the present invention, in another aspect, The present invention relates to an antiviral composition for HCV containing a salt as an active ingredient.

The term "pharmaceutically acceptable salt" in the present invention refers to any salt of the compound of the present invention that does not exhibit toxicity or is unsuitable for pharmaceutical use while retaining the biological properties of the compound of the present invention. Such salts may be derived from, and include, various organic and inorganic counterions known in the art. Such salts include (1) organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, But are not limited to, tartaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- Examples of the acid addition salts include acid addition salts such as phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butyl acetic acid, , Benzoic acid, glutamic acid, hydroxynaphthoic acid, Acid addition salts formed as florisil acid, stearic acid, sulfamic acid cyclohexylsulfonyl, kwinsan, mu acid; Or (2) the acidic proton present in the parent compound is selected from the group consisting of (a) metal ions (e.g., alkali metal ions, alkaline earth metal ions or aluminum ions), alkali metal or alkaline earth metal hydroxides (e.g., sodium, potassium, calcium, magnesium (B) an organic base such as an aliphatic, alicyclic or aromatic organic amine such as ammonia, methylamine, dimethylamine, diethylamine, picoline , Ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylene-diamine, chloropropane, diethanolamine, procaine, N- Amine, N-methylglucamine piperazine, tris (hydroxymethyl) -aminomethane, salts formed when coordinating with tetramethylammonium hydroxide, and the like.

Examples of salts include sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium salts. When the compound contains a basic functional group, salts with non-toxic organic acids or inorganic acids such as hydrohalides (for example, Chloride, or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentyl propionate, glycolate, glutarate, (4-hydroxybenzoyl) benzoate, or a salt thereof, such as maltodextrin, maltodextrin, maltodextrin, maltodextrin, maltodextrin, Methanesulfonate mesylate), ethanesulfonate (e.g., methanesulfonate, methanesulfonate, methanesulfonate, Benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, benzenesulfonate, 2-hydroxybenzenesulfonate, Methylbicyclo [2.2.2] -oct-2-en-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, laurylsulfate Phosphate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexyl sulfamate, quinate, muconate salts and the like.

The ligase tip or pharmaceutically acceptable salt thereof is administered to a subject or patient wherein the "subject (s)" is an animal, such as a non-primate (e.g., cow, pig, Mammals including, for example, dogs, rats and mice) and primates (e.g., monkeys such as cynomolgous monkeys, chimpanzees and humans) and, for example, humans. In one embodiment, the subject is resistant or unresponsive to current treatment for hepatitis C infection. In another embodiment, the subject is an animal for livestock (e. G., Horse, cattle, pig, etc.) or a pet animal (e. In a preferred embodiment, the subject is a human.

The term "therapeutic agent (s) or therapeutic composition" as used herein refers to any agent (s) that can be used to treat or prevent a disorder or one or more indications thereof. In certain embodiments, the term "therapeutic agent" refers to a ligase tip that is a compound of the present invention. In another particular embodiment, the term "therapeutic agent" does not refer to a compound of the invention. In one embodiment, the therapeutic agent is an agent that is known to be useful for treating or preventing a disorder or one or more of its symptoms, or that has been or has been used for that purpose.

The glyceride tip used in the process of the invention is preferably prepared by using the compound of formula I, if appropriate in salt form, alone or in combination with one or more suitable pharmaceutical acceptable carriers such as diluents or adjuvants, Or in combination with another anti-HCV agent.

In clinical practice, the derivatives of the present invention may be administered by any conventional route, particularly an oral, parenteral, rectal route, or by inhalation (e.g., in the form of an aerosol).

As the solid composition for oral administration, tablets, pills, hard gelatin capsules, powders or granules can be used. In these compositions, the compounds according to the invention are mixed with one or more inert diluents or adjuvants such as sucrose, lactose or starch. These compositions may contain, in addition to diluents, materials such as, for example, lubricants such as magnesium stearate, or coatings for controlled release.

As a liquid composition for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing an inert diluent such as water or liquid paraffin can be used. These compositions may also contain, in addition to diluents, substances such as, for example, wetting agents, sweeteners or flavoring agents.

The composition for parenteral administration may be an emulsion or a sterile solution. As the solvent or the vehicle, propylene glycol, polyethylene glycol, vegetable oil, in particular olive oil or injected organic ester such as ethyl oleate can be used. These compositions may also contain adjuvants, in particular wetting agents, isotonizing agents, emulsifying agents, dispersing agents and stabilizers. Sterilization can be performed in a number of ways, for example, by using a filter for bacteria, irradiating radiation, or heating. They can also be prepared in the form of sterile solid compositions which, when in use, can be dissolved in sterile water or any other injectable sterile medium.

Compositions for rectal administration are capsules for suppository or rectal administration and they contain excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols in addition to the active ingredients.

The composition may also be an aerosol. When used in the form of a liquid aerosol, the composition may be a stable sterile solution or may be a solid composition which, when in use, is dissolved in non-sterile sterile water, saline or any other pharmaceutically acceptable vehicle. When used in anhydrous aerosol form for direct inhalation, the active ingredient is finely divided and mixed with a water soluble solid diluent or vehicle, such as dextran, mannitol or lactose.

In one embodiment, the compositions of the invention are in the form of pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms of the present invention may contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound of the invention, or other prophylactic or therapeutic agent), and typically one or more pharmaceutically acceptable Carrier or excipient.

The term "pharmaceutically acceptable" in certain embodiments and contexts means that it is listed in the pharmacopoeia which has been approved by the government or a regulatory agency of the government as being capable of being used in animals, and more particularly in humans, do. The term "carrier" refers to a diluent, such as a diluent, such as Freund ' s adjuvant (complete and incomplete adjuvant), excipient or vehicle, which is administered with the therapeutic. Such pharmaceutical carriers may be sterile liquids such as water, and oils, including oils or synthetic oils, of petroleum, animal or vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.

Saline solution, and dextrose and glycerol aqueous solutions can also be used as a liquid carrier, especially a injectable solution. An example of a suitable pharmaceutical carrier is described in "Remington ' s Pharmaceutical Sciences" by E. W. Martin.

Conventional pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well known to those skilled in the pharmaceutical arts and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, hulls, silica gel, sodium stearate, glycerol monostearate, Talc, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form will depend upon a variety of factors known in the art including the manner in which the dosage form is administered to the subject and the particular active ingredient involved in the dosage form, Optionally, the composition or single unit dosage form may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The present invention also encompasses a pharmaceutical composition and dosage form comprising the active ingredient. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical industry as a means of long term storage to determine the properties of the formulation (e.g., shelf life or stability) over time. See, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379 < / RTI > In fact, water and heat accelerate the decomposition of some compounds. Thus, the effects of water on the formulation can be very important, as water and / or moisture are typically generated during manufacture, handling, packaging, storage, transport and use of the formulation.

The anhydrous pharmaceutical compositions and dosage forms of the present invention may be prepared using anhydrous or low moisture content and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms comprising one or more active ingredients, including lactose, and primary or secondary amines, are contemplated to be in the form of an anhydrous form if they are expected to come into substantial contact with moisture and / or moisture during manufacture, packaging and / . The anhydrous pharmaceutical composition should be prepared and stored so that its anhydrous properties are maintained. It is therefore desirable to prevent the anhydrous composition from being exposed to water and to be packaged using known materials to be included in a suitable standard kit. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (e.g. vials), blister packs and strip packs.

The invention also encompasses pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of degradation of the active ingredient. Such compounds (referred to herein as "stabilizers") include, but are not limited to, antioxidants (such as ascorbic acid), pH buffers or salt buffers.

The pharmaceutical compositions and single unit dosage forms may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Formulations for oral administration may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, in a suitable amount and in a form suitable for administration to a subject. The formulation should be appropriate for the mode of administration. In a preferred embodiment, the pharmaceutical composition or single unit dosage form is sterile and is in a form suitable for administration to a subject, preferably an animal subject, more preferably a mammalian subject, most preferably a human subject.

The pharmaceutical composition of the present invention is formulated to be suitable for its intended route of administration. Examples of routes of administration include parenteral administration routes such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, oral, sublingual, inhalation, intranasal, transdermal, topical, intratumoral, Rectal routes of administration, but are not limited thereto. In certain embodiments, the composition is formulated into a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to a human according to conventional procedures. In one embodiment, the pharmaceutical composition is formulated into a composition that is subcutaneously administered to a human according to conventional procedures.

Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If desired, the composition may also contain a solubilizer and a local anesthetic (e.g., lignocaine) to soothe the pain at the injection site.

Examples of dosage forms include tablets; Caplet; Capsules such as soft elastic gelatin capsules; Casse; Troches; Rosenji; A dispersion agent; Suppository; Ointment; Poultice (poultice); Paste; Powder; dressing; cream; salve; Solution agent; patch; Aerosols (e. G., Nasal sprays or inhalers); Gel; Liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (e. G., Aqueous or nonaqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions and elixirs; A liquid dosage form suitable for parenteral administration to a subject; And sterile solids (e.g., crystalline or amorphous solids) that can provide a liquid dosage form suitable for parenteral administration to a subject when reconstituted.

The types of compositions, forms and dosage forms of the present invention will typically vary depending on their use. For example, the dosage form used for the initial treatment of a viral infection may contain more than one active ingredient in an amount greater than that contained in the dosage form used for the maintenance therapy of the viral infection. Similarly, parenteral dosage forms may contain one or more active ingredients in an amount that is less than that contained in the oral dosage form used in the same disease or disorder treatment. Such methods and other ways of modifying certain dosage forms involved in the present invention will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

The ingredients of the compositions of the invention are generally anhydrous lyophilized powder or water free concentrates in sealed containers such as, for example, ampoules or chasers indicating the amount of active agent, either separately or mixed together . When the composition is administered by injection, the composition may be dispensed into an injection vessel containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, it may be provided with injectable sterile water or saline ampoules so that the ingredients may be mixed prior to administration.

Typical dosage forms of the invention comprise a compound of the present invention, ligoseride, or a pharmaceutically acceptable salt, solvate or hydrate thereof in an amount ranging from about 0.1 mg to about 1000 mg / day, Administered once a day, preferably once a day, with the meal.

The pharmaceutical compositions of the invention suitable for oral administration may be presented in separate dosage forms such as tablets (e.g., chewing tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain a predetermined amount of the active ingredient and may be prepared by the preparation methods well known to those skilled in the art. Generally, see Remington ' s Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). In certain embodiments, the oral dosage form is a solid and is prepared under anhydrous conditions using anhydrous ingredients as described in detail in the paragraph above.

However, the scope of the present invention extends to other than anhydrous solid oral dosage forms. Conventional oral dosage forms of the present invention are prepared by combining the active ingredient (s) with one or more excipients in intimate admixture according to conventional pharmaceutical synthetic techniques. The excipient can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral or parenteral dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives and coloring agents. Suitable excipients for use in solid oral dosage forms (e.g., powders, tablets, capsules and caplets) include, but are not limited to, starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants . When solid excipients are used, tablets and capsules are the most advantageous oral dosage unit forms because they are easy to administer. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by any formulation method.

In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredient with a liquid carrier, finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired shape. For example, tablets may be prepared by compression or molding. Compressed tablets may optionally be prepared by compressing the active ingredient in a free-flowing form (e. G., Powder or granules) mixed with excipients in a suitable machine. Molded tablets may be prepared by molding a mixture of the powdered compound soaked with an inert liquid diluent in a suitable machine.

Examples of excipients that may be used in the oral dosage forms of the present invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Suitable binders for use in pharmaceutical compositions and dosage forms include corn starch, potato starch or other starch, gelatin, natural and synthetic gums such as acacia gum, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, Cellulose and derivatives thereof (for example, ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropylmethyl cellulose, , No. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch , Pregelatinized starch, and mixtures thereof, but are not limited thereto. Binders or fillers included in the pharmaceutical compositions of the invention are typically present in an amount of from about 50% to about 99% by weight of the pharmaceutical composition or dosage form. Suitable forms of microcrystalline cellulose include, but are not limited to, materials available from Avicel PH 101, Avicel PH 103, Avicel RC 581, Avicel PH 105 (FMC Corporation, American Viscose Division, Avicel Sales, (Available from Markus Hook, Pa., USA), and mixtures thereof.

The use of a disintegrant in the composition of the present invention provides a tablet that disintegrates when exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate in storage, and tablets containing too little disintegrant may not disintegrate at the desired rate or may not disintegrate under the desired conditions. Thus, a solid oral dosage form of the invention should be formed using a sufficient amount of a disintegrant that is neither too much nor too little and does not deleteriously alter the release of the active ingredient. The amount of disintegrant used will depend on the type of formulation and can be readily determined by those skilled in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent, specifically from about 1 to about 5 weight percent of a disintegrant. Disintegrants that may be used in the pharmaceutical compositions and dosage forms of the invention include agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, But are not limited to, pregelatinized starches, other starches, clays, other alginates, other celluloses, gums, and mixtures thereof.

Lubricants that may be used in the pharmaceutical compositions and dosage forms of the invention include calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycols, other glycols, stearic acid, sodium lauryl sulfate , Talc, hydrogenated vegetable oils (such as peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, But are not limited thereto. Additional lubricants include, for example, ciloid silica gels (AEROSIL 200, manufactured by WR Grace Co., Baltimore, MD), solidified aerosols of synthetic silica (Degussa Corporation (Commercially available from Degussa Co., Piano, TX), CAB O SIL (exothermic silicon dioxide product, available from Cabot Co .; Boston, MA), and mixtures thereof . When used, lubricants are typically used in amounts of less than about 1% by weight of the pharmaceutical composition or dosage form into which they are incorporated.

The liposome tip of the present invention can be administered, for example, by controlled release means or delivery devices known to those skilled in the art. Such dosage forms can be formulated for administration by any convenient means, including, for example, by means of hydropropylmethylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayered coatings, microparticles, liposomes, microspheres, Water can be used to retard release or controlled release of one or more active ingredients. Suitable controlled release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the active ingredients of the present invention. Accordingly, the present invention encompasses single unit dosage forms suitable for oral administration, such as tablets, capsules, gel caps, caplets and the like, which are modified into controlled release formulations.

All controlled release drugs have a common purpose to provide improved drug therapy over that achieved by their corresponding non-controlled release drugs. Ideally, the use of a controlled release formulation that is most appropriately designed for medical treatment is characterized by the use of a minimal amount of drug substance to cure or control symptoms within a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, reduced administration frequency and increased compliance of the subject. In addition, controlled release formulations may be used to affect the onset of action or other characteristics of the drug, such as the blood concentration of the drug, which may also have an adverse effect (adverse effect).

Most controlled-release formulations initially release a quantity of the drug (active ingredient) immediately indicative of the desired therapeutic effect, and release the drug in a stepwise and sustained manner over a prolonged period of time to maintain the therapeutic or prophylactic effect level ≪ / RTI > Thus, to maintain a constant level of drug in the body, the drug must be released from the dosage form at a rate that is metabolized and replaces the amount of drug secreted from the body. Controlled release of the active ingredient may be stimulated by a variety of conditions, including pH, temperature, enzyme, water, or other physiological conditions or compounds.

In certain embodiments, the drug may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, a liposome, or other mode of administration. In one embodiment, a pump may be used (Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, a polymeric material may be used. In another embodiment, the controlled-release system can be located at a suitable site as determined by a person skilled in the art, i.e. only a fraction of the systemic dose is required (see, for example, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)). Other controlled release systems have been reviewed and discussed by Langer (Science 249: 1527-1533 (1990)). The active ingredient may be a solid internal matrix such as, for example, polymethyl methacrylate, polybutyl methacrylate, plasticized or non-digested polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene , Hydrogels of polyesters such as polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate, copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as esters of acrylic acid and methacrylic acid, collagen, Such as polyethylene, polypropylene, an ethylene / propylene copolymer, an ethylene / ethyl acrylate copolymer, an ethylene / vinyl acetate copolymer, an ethylene / vinyl acetate copolymer, Acetate copolymer, silicone rubber, polydimethylsiloxane, Friendly rubber, chlorinated polyethylene, polyvinyl chloride; Vinyl acetate copolymers, ethylene / vinyl acetate / vinyl alcohol terpolymers, ethylene / vinyl acetate copolymers, ethylene / vinyl acetate copolymers, ethylene / vinyl acetate copolymers, ethylene / vinyl acetate copolymers, ethylene / vinyl acetate copolymers, Vinyloxyethanol copolymer, which is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in the release rate control step. The proportion of the active ingredient in such a parenteral composition is highly dependent on the specific properties as well as the needs of the subject.

The present invention also provides parenteral dosage forms. Parenteral dosage forms may be administered to the subject by a variety of routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial routes. Because these parenteral routes of administration typically avoid the natural defense pathway of the subject to contaminants, it is preferred that the parenteral dosage form be sterile or sterile prior to administration to the subject. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, anhydrous products that may be dissolved or suspended in a pharmaceutically acceptable injectable vehicle, injectable suspensions, and emulsions.

Suitable vehicles which can be used to provide parenteral dosage forms of the invention are known to those skilled in the art. Examples thereof include water for injection (USP); Aqueous vehicles such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer injection; Water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; And non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. In addition, compounds that increase the solubility of one or more of the active ingredients disclosed herein may also be incorporated into the parenteral dosage forms of the invention.

The present invention also provides transdermal, topical and mucosal dosage forms. The transdermal, topical and mucosal administration forms of the present invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000) and Introduction to Pharmaceutical dosage form, 4th ed., Lea & )].

Dosage forms suitable for the treatment of oral mucosal tissues may be formulated with a solution for oral cleaning or a gel for oral use. The transdermal dosage form may also include a "storage small" or "matrix" patch, To the site for a specified period of time to infiltrate the desired amount of active ingredient.

Suitable excipients (e.g., carriers and diluents) included in the present invention, and other materials that can be used to provide transdermal, topical and mucosal dosage forms, are well known to those skilled in the pharmaceutical arts, Depending on the particular organization to which it applies. In view of this fact, typical excipients include water to form lotions, tinctures, creams, emulsions, gels or ointments, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl Palmitates, mineral oils, and mixtures thereof, which are non-toxic and pharmaceutically acceptable. If desired, a moisturizing or wetting agent may be added to the pharmaceutical composition and dosage form. Examples of such additional ingredients are known in the art. See, for example, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000).

Depending on the particular tissue to be treated, additional ingredients may be used before treatment with the active ingredient of the invention, with the active ingredient of the present invention, or after treatment with the active ingredient of the present invention. For example, penetration enhancers can be used to assist in delivering the active ingredient to the tissue. Suitable penetration enhancers include acetone; Various alcohols such as ethanol, oleyl and tetrahydrofuryl; Alkyl sulfoxides such as dimethyl sulfoxide; Dimethylacetamide; Dimethylformamide; Polyethylene glycol; Pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (povidone, polybidone); Urea; And various water soluble or water insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

In addition, the pH of the pharmaceutical composition or dosage form, or the pH of the tissue to which the pharmaceutical composition or dosage form is applied, can be adjusted to improve the delivery of one or more active ingredients. Similarly, the transfer can be improved by adjusting the polarity of the solvent carrier, its ionic strength or its tautness. A compound, such as a stearate, may be added to a pharmaceutical composition or dosage form to improve the delivery by advantageously altering the hydrophilicity or lipophilicity of the one or more active ingredients. In this regard, the stearate may serve as a liquid vehicle for formulation, an emulsifier or a surfactant, and as a delivery enhancer or penetration enhancer. Other salts, hydrates or solvates of the active ingredient may further adjust the properties of the resulting composition.

When treating humans, the physician will determine the dosages that are considered most appropriate depending on the preventative or curative treatment, and the age, weight, stage of infection and other factors specific to the subject being treated. Generally, in the case of an adult, the dosage is about 1 to about 1000 mg / day, about 5 to about 250 mg / day, or about 10 to 50 mg / day. In certain embodiments, the dosage is from about 5 to about 400 mg / day, more preferably from 25 to 200 mg / day per adult. An administration rate of about 50 to about 500 mg / day is also preferred.

In a further aspect, the present invention provides a method for treating or preventing hepatitis C virus infection comprising administering to a subject in need thereof an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, which has a high therapeutic index against hepatitis C virus, Lt; RTI ID = 0.0 > hepatitis C < / RTI > infection. The therapeutic index can be determined according to any method known to a person skilled in the art, for example, the method described in the following examples. In certain embodiments, the therapeutic index is a ratio of the concentration of the compound exhibiting toxicity to the concentration of the compound effective against hepatitis C virus. Toxicity can be measured by any technique known to those skilled in the art, including cytotoxicity (e.g., IC50 or IC90) and lethal dose (e.g., LD50 or LD90). Likewise, effective concentrations can be determined using any technique known to those skilled in the art, including effective concentrations (e.g., EC50 or EC90) and effective dosages (e.g., ED50 or ED90). Preferably, comparable measurements are compared (e.g., IC50 / EC50, IC90 / EC90, LD50 / ED50 or LD90 / ED90). In certain embodiments, the therapeutic index may be as high as 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0 or more.

The amount of a compound or composition of the invention effective to prevent, treat, manage or ameliorate a disorder or one or more symptoms thereof will vary depending on the nature and severity of the disease or condition, and the route by which the active ingredient is to be administered. The frequency and dosage of administration will also depend on the particular therapeutic agent (e.g., therapeutic or prophylactic) administered, the severity of the disorder, disease or condition, the route of administration, as well as the age, weight, It will vary depending on the specific factors. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Examples of dosage amounts of the composition include, for example, from about 10 μg / kg to about 50 mg / kg, from about 100 μg / kg to about 25 mg / kg, as the amount of active compound (mg or μg) kg, or about 100 [mu] g / kg to about 10 mg / kg.

For compositions of the invention, the dosage administered to a subject is typically from 0.140 mg to 3 mg per kilogram body weight of the subject based on the weight of the active compound. Preferably, the dose administered to the subject is from 0.20 mg to 2.00 mg or 0.30 mg to 1.50 mg per kg body weight of the subject.

In general, the daily dosage range of the compositions of the present invention proposed for the symptoms described herein is from about 0.1 to about 1000 mg / day, which is administered once a day, or divided doses for a day. In one embodiment, the daily dose is divided into equivalent amounts and administered twice daily. The daily dosage range should be specifically from about 10 to about 200 mg / day, more particularly from about 10 to about 150 mg / day, and even more specifically from about 25 to about 100 mg / day. As will be apparent to those skilled in the art, in some cases it may be necessary to use the active ingredient in dosages outside of the dosage ranges set forth herein. In addition, the clinician or therapist will notice how and when to stop, adjust, or terminate the therapy in relation to the subject's response.

As is well known to those skilled in the art, different therapeutically effective amounts can be applied for different diseases and conditions. Similarly, an amount sufficient to prevent, manage, treat, or ameliorate the disorder, but insufficient to cause side effects associated with the composition of the present invention, or sufficient to reduce the side effects, is also included in the dosage and dosage schedule design described above do. Also, when multiple doses of the composition of the present invention are administered to a subject, not all doses need to be the same. For example, the dosage administered to a subject can be increased to improve the prophylactic or therapeutic effect of the composition, which can be reduced to reduce one or more side effects experienced by a particular subject.

The lyoglocer tip or a pharmaceutically acceptable salt thereof of the present invention can be used for the amelioration or prevention of liver disease caused by hepatitis C virus, and the present invention thus provides, from another viewpoint, To a health functional food composition for improving or preventing liver disease caused by HCV containing an acceptable salt as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and improving liver diseases caused by HCV.

The term " health functional food "used in the present invention refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on health functional foods, To be used for the purpose of obtaining a beneficial effect for health use such as controlling nutrients or physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture obtained by mixing the active ingredient of the present invention, glyceride, with an excipient, a binder, a disintegrant and other additives in a usual manner, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a mixture of a hard capsule of the present invention with an additive such as an excipient, which is an effective ingredient of the present invention. The soft capsule is prepared by mixing the above- And the like can be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the compound of formula (1), the active ingredient of the present invention, excipient, binder, disintegrant and the like by a conventionally known method, It may be applied to the skin, or the surface may be coated with a substance such as starch or talc.

The granular health functional food may be prepared by granulating a mixture of Lysozyme tip, which is an effective ingredient of the present invention, with excipient, binder, disintegrant, etc., by a conventionally known method and, if necessary, adding a flavoring agent, And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

ELAVL1 / On Hur  by miRNA -122 Control and Hepatitis C Bias Replication Correlation

We have searched intracellular molecules that bind miRNA-122, which is known to play an essential role in the translation / replication / proliferation of hepatitis C virus (Jopling et al.). In this study, the intracellular molecules bound to biotinylated miRNA-122 were analyzed by LC-MS / MS technique. As a result, specific binding between miRNA-122 and ELAVL1 / HuR was observed, ELAVL1 / HuR removal To monitor transcription / replication of HCV during knockout, the following experiments were performed.

1-1: ELAVL1 / HuR and miRNA - 122  Identification of specific binding

There are four kinds of isomiR for miRNA-122 in liver cells (FIG. 1A). Specific binding between miRNA-122 and miRNA-122 was confirmed by REMSA (RNA electro-mobility shift assay) and specific recombinant ELAVL1 / HuR And the REMSA process are as follows.

Escherichia coli HuR (GST-HuR) fused with recombinant glutathione S -transferase (GST) from plasmid pGEX-KG-HuR (WT) using strain BL21 (DE3) pLysS (Promega). GST-HuR protein expression was induced at OD 600 0.5 by adding isopropyl -? - D-thiogalactopyranoside (final concentration 0.5 mM). After incubation at 25 ° C for 5 hours, the cells were harvested and lysed in lysis buffer [20 mM phosphate (pH 7.6), 300 mM NaCl, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM β-mercaptoethanol and 10% glycerol (v / v)] and then subjected to ultrasonic disintegration treatment. The resulting cell extract was loaded onto Glutathione agarose 4B (Peptron, Dajeon, Korea), washed with lysis buffer, and bound GST-HuR was eluted with reducing glutathione. Purified recombinant GST-HuR was carried out in a dialysis buffer containing 20 mM HEPES-KOH (pH 7.5 ), 50 mM KCl, 5 mM MgCl 2, 10% glycerol (v / v). For REMSA, the 5 'end of artificially synthesized miRNA-122 was labeled with ³² P using T4 polynucleotide kinase (PNK) and 25,000 cpm was used for each reaction. Each recombinant protein was mixed with 1 mM MgCl ₂ , 5% glycerol (v / v), 0.5 mM dithiothreitol, 1 unit / μl RNaseOUT, reacted at 30 ° C for 20 minutes, 5% non-denaturing gel and the results were analyzed by autoradiography.

As a result, the wild type showed only one nucleotide difference in the 3 'terminal region of miRNA-122, and the binding was the strongest, indicating that the 3' terminal region of miRNA-122 is very important for binding to ELAVL1 / HuR. Figure IB shows that ELAVL1 / HuR is directly associated with miRNA-122, which accounts for 70% of human hepatocyte miRNA and is believed to play an indispensable role in the replication process of hepatitis C virus.

1-2: ELAVL1 / Hur and  Identification of Hepatitis C Bias Replication Correlation

In order to investigate the correlation between ELAVL1 / HuR and hepatitis C virus replication, a luciferase-expressing hepatitis C subgenomic replicon derived from the infectious hepatitis C virus production system (pJFH1) was introduced and the CRISPR / Cas9 system was applied. ELAVL1 / HuR When ELAVL1 / HuR was knocked out using specific sgRNA, the degree of synthesis of hepatitis C virus and the activity of the hepatitis C subgenomic replicon capable of luciferase assay were tracked. When ELAVL1 / HuR was knocked out, The inhibition of translation / replication of hepatitis subgenomic replicon was confirmed as follows.

Knockout using the CRISPR / Cas9 system of the target gene (ELAVL1 / HuR) was performed using pLentiCRISPRv2 (Addgene) and stably transfected Huh7 cells were selected using puromycin. Cells were selected with puromycin and collected. Colonies of selected Huh7 cells were analyzed by Western blotting 30 days after infection. The genomic DNA of the selected colonies was isolated using a genomic DNA column kit (Zymo Reserch), amplified from the genomic DNA adjacent to the ELAVL1 / HuR target site for the inserted guide RNA sequence, and the resulting amplicon sequence was subjected to DNA sequencing Genentech).

First, the rear one or two kinds of loose fur raised assay is cut into subgenomic replicon of pSGR-JFG1 / Luc, or mutant chain pSGR-JFG1 / Luc-GND restriction enzyme (Ralf Bartenschlager) of the active site of the NS5B available in vitro transcription To synthesize the RNA of the hepatitis C subgenomic replicon expressing Luciferase. The cells were transfected with ELAVL1 / HuR knockout cells using naive Huh7 cell line or CRISPR / Cas9 gene scissors by electroporation (1 ug of RNA into 1x10 ^ 6 cells) After transfection, luciferase assay was performed by fractionation at each time. The fractionated cells were lysed with 1 × passive lysis buffer (Promega), and a portion of the lysate was analyzed by Dual-Luciferase® Reporter Assay System (Promega). Normalization of the firefly luciferase signal with Renilla signal or total protein amount was performed

As a result, as shown in FIG. 1C, when ELAVL1 / HuR was KO, the hepatitis C subgenomic replicon translation / replication was inhibited.

In addition, Western blotting was performed. First, anti-HuR antibody (mouse monoclonal antibody, 3A2, Santa Cruz Biotech), anti-eIF-4GI (rabbit polyclonal antibody, Pohang University of Science and Technology), anti-GST antibody (rabbit polyclone, Genescript) . For Western blot analysis, the cell lysate was mixed with 5 × protein sample buffer, denatured at 95 ° C. for 5 minutes, and electrophoresed on an 11% polyacrylamide-SDS gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 4 ° C and 80v for 1 hour. After 5% non-fat milk in TBS (50 mM Tris-HCl [pH 7.4] and 150 mM NaCl) containing 0.1% Tween-20 was reacted at room temperature for 30 minutes and then reacted with primary antibody overnight at 4 ° C . The nitrocellulose membrane was washed three times with TBS-T for 15 min before treatment with secondary antibody. Anti-rabbit and anti-mouse IgG conjugated with horseradish peroxidase (1/5000 dilution, Vector Laboratories) was used as a secondary antibody.

As a result, when ELAVL1 / HuR was KO as shown in Fig. 1D, the hepatitis C subgenomic replicon translation / replication was completely inhibited.

HCV  As an inhibitor Rigoser Tip  Separation and analysis

2-1: HCV  As an inhibitor Rigoser Tip  detach

To validate the importance of cellular signaling in the interaction of HCV's intracellular molecules and to investigate the effects of these processes on the translation / replication / proliferation of HCV, we used a subgenomic replicon of HCV that can be assayed for reporter And a primary screening was performed using an inhibitor library for various kinases in cells.

The Huh7 cell line containing the assayable HCV 1b subgenomic replicon HIRL-2Aneo-NSrep was plated and treated with 1 μM Selleckchem cellular kinase inhibitory chemicals (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). After 24 hours, Luciferase assay Respectively. The fractionated cells were lysed with 1 × passive lysis buffer (Promega), and a portion of the lysate was analyzed by Dual-Luciferase® Reporter Assay System (Promega). Normalization of the firefly luciferase signal with Renilla signal or total protein amount was performed

As a result, the first screening for 211 Selleckchem cellular kinase inhibitory chemicals showed that a new inhibitor of translation / proliferation of HCV, rigosertib (# 65, Fig. 2C) . Furthermore, this substance strongly inhibited replicon proliferation similarly to danoprevir, which was developed by Roche in clinical use, and showed an inhibitory effect of 80% or more even at a low concentration (0.2 μM) (FIG. 2A).

2-2: HCV  As an inhibitor Rigoser Tip  Analysis pulldown  assay)

The isolated recombinant GST protein or GST-HuR protein was diluted with Drosha lysis buffer [20 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.2 mM EDTA, 2 mM MgCl ₂ , 2 mM CaCl ₂ , protease inhibitor cocktail] Followed by addition of RNase inhibitor and mixing with biotinylated-rigosertib (FIG. 2D) combined with streptavidin-magnetic bead (Promega). After binding, the bead sample was washed 4 times with the modified Drosha wash buffer and the residual wash buffer was completely removed. The 5x sample buffer for western blotting was then added to the beads and Western blotting was performed using GST-specific antibodies (Genescript).

As a result, as shown in FIG. 2B, it was confirmed that the molecule ELAVL1 / HuR, which is found to be important for HCV translation and replication, directly binds to lysophosphorylase and lysoserthide plays an important role in the replication process of HCV translation Replication / proliferation of HCV by inhibiting the direct binding of miRNA-122, a molecule known to be involved, to the ELAVL1 / HuR molecule, an intracellular molecule (Fig. 2E).

Rigoser Tip Anti-HCV  Revalidation of effects

In order to re-verify that the glyceride tip identified in Example 2 is a specific and effective inhibitor of HCV infection, the following experiment was further performed.

First, an assayable HCV 1b subgenomic replicon HIRL-2Aneo-NSrep and a firefly luciferase were assayed against an HCV-specific inhibitor ligase serum extracted using a subgenomic replicon of HCV capable of a reporter assay on the same line as the screening result of Example 2 The Huh7 cell line was plated in 24 wells at 2 × 10 ^ 4, and 1 μM rigosertib was treated for 0, 24, 48, and 72 hours and then fractionated at each time point to perform luciferase assay. The fractionated cells were lysed with 1 × passive lysis buffer (Promega), and a portion of the lysate was analyzed with Dual-Luciferase® Reporter Assay System (Promega). Normalization of the Renilla signal was performed using a firefly luciferase signal.

As a result, it was confirmed that the translation / proliferation of HCV was inhibited as shown in Fig. 3A. Further, when the same experiment was conducted and the serum tip was treated at a concentration of 0.5 μM, it was confirmed that the inhibition was the same as shown in FIG. 3B.

In contrast, the inhibitory mechanism of the liposome tip according to the present invention inhibits the action of a host factor (a human hepatocyte molecule) rather than a protein produced by HCV itself, unlike the previously developed anti-HCV therapeutic agent (miRNA- 122 and ELAVL1 / HuR), which do not target HCV proteins, are anticipated to be a new concept anti-hepatitis C virus agent that can solve the existing drug resistance problems. It is anticipated to be a good treatment strategy for HCV that is used alone or in combination with ribavirin, which is known to be inexpensive and resistant to HCV.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Unsigned

<110> National Cancer Center <120> Novel Use of Rigosertib for treating Hepatitis C Virus Disease <130> P12661 <150> KR 10-2017-0031850 <151> 2017-03-14 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Wild type of miR-122 <400> 1 uggaguguga caaugguguu ug 22 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Isomir of miR-122 (+ A) <400> 2 uggaguguga caaugguguu uga 23 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Isomir of miR-122 (+ U) <400> 3 uggaguguga caaugguguu ugu 23 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Isomir of miR-122 (-G) <400> 4 uggaguguga caaugguguu u 21 <210> 5 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-21 <400> 5 uagcuuauca gacugauguu ga 22

Claims (7)

A pharmaceutical composition for the treatment or prevention of liver disease caused by hepatitis C virus (HCV), which comprises lyogosertib or a pharmaceutically acceptable salt thereof as an active ingredient. The method according to claim 1, wherein the liver disease caused by hepatitis C virus is selected from the group consisting of hepatitis C, hepatic fibrosis caused by hepatitis C virus, hepatitis C virus, and hepatitis C virus Or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, which is administered in combination with an HCV therapeutic agent or an antiviral agent against HCV. The pharmaceutical composition according to claim 1, wherein the ligase tip binds to ELAVL1 / HuR to inhibit binding of miRNA-122 to ELAVL1 / HuR. An antiviral composition for HCV comprising lysogliptin or a pharmaceutically acceptable salt thereof as an active ingredient. A health functional food composition for the improvement or prevention of liver disease caused by HCV, which contains Lysozyme tip or a pharmaceutically acceptable salt thereof as an active ingredient. The method according to claim 6, wherein the liver disease caused by the hepatitis C virus is selected from the group consisting of hepatitis C, liver fibrosis caused by hepatitis C virus, hepatitis C virus, and liver cancer caused by hepatitis C virus Or a pharmaceutically acceptable salt thereof.

Images