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WO2018169283A1 - Nouvelle utilisation du rigosertib pour le traitement d'une maladie causée par le virus de l'hépatite c - Google Patents

Nouvelle utilisation du rigosertib pour le traitement d'une maladie causée par le virus de l'hépatite c Download PDF

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Publication number
WO2018169283A1
WO2018169283A1 PCT/KR2018/002938 KR2018002938W WO2018169283A1 WO 2018169283 A1 WO2018169283 A1 WO 2018169283A1 KR 2018002938 W KR2018002938 W KR 2018002938W WO 2018169283 A1 WO2018169283 A1 WO 2018169283A1
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Prior art keywords
hepatitis
virus
hcv
rigosertib
hur
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PCT/KR2018/002938
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English (en)
Korean (ko)
Inventor
김종헌
박종배
조성찬
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National Cancer Center Japan
National Cancer Center Korea
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National Cancer Center Japan
National Cancer Center Korea
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a novel use of ligosertip, and more particularly, to the use or prevention of ligosertip for the prevention or treatment of liver disease by hepatitis C virus and the antiviral use against hepatitis C virus.
  • Rigosertib (Onconova Therapeutics, Inc.) is a synthetic benzyl styryl sulfone substance, previously known as an anticancer agent, and has effects such as treatment of proliferative diseases (US7598232), myelodysplastic syndrome and acute myeloid leukemia (US8664272). Is known. However, there have been no reported uses associated with liver disease and further hepatitis C.
  • Drug repositioning is a concept that develops new indications for drugs currently in use and is recognized as an alternative to dramatically improve the potential for new drug development.
  • New drug re-creation can reduce the cost and duration of new drug development if the safety of the candidate drug is verified and validated.
  • Drug re-creation has (1) the use, preparation and stability of the drug (Drug) established; (2) Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) data, namely drug absorption, diffusion, metabolism, excretion and drug toxicity information, are established; (3) the probability of failure due to safety issues is significantly lower, based on the results of clinical trials of the drug prior to drug use change; (4) Post-marketing Surveillance (PMS) has the advantage of not having to spend time obtaining safety data.
  • Drug Absorption, Distribution, Metabolism, Excretion and Toxicity
  • hepatitis C is mostly symptomatic in the early stage of infection, and when hepatitis C is infected, it is known to become chronic in more than 75% of the hepatitis B virus infection.
  • Chronic hepatitis C is a disease in which the liver's inflammation continues chronically when the body's immune system does not completely remove the hepatitis C virus and the infection continues for more than 6 months.
  • Hepatitis C virus is an RNA virus classified as the hepacivirus genus of the Flaviviridae family, and was discovered in the United States in 1989. It is a clinically important virus that causes hepatitis, cirrohosis and hepatocellular carcinoma (HCC). HCV is the leading cause of chronic hepatitis in developed countries such as the United States and Europe. According to the WHO, hepatitis C virus (HCV) is a more serious problem, with approximately 200 million people worldwide, more than 3% of the total population, and an increase of 3-4 million additional infections each year. .
  • liver cirrhosis progress to liver cancer at an incidence of 1-5% per year. It is known to become. About 12% of liver cirrhosis patients and about 15% of hepatocellular carcinoma patients are caused by hepatitis C virus and are known to be the main cause of chronic liver disease with hepatitis B virus.
  • HCV high-density lipoprotein
  • peginterferon and ribavirin the most widely used therapeutic agent for HCV
  • the treatment rate is only 30-50%, which is also effective depending on the genotype of HCV.
  • therapies targeting HCV non-structural proteins such as Sovaldi and Harvoni of Gilead Sciences, have been reported, but these are expensive to apply to the treatment of most patients.
  • the present inventors have made an effort to develop a new HCV infection-treated or preventive agent that can solve the problem of resistance of existing drugs while acting specifically and effectively against HCV infection, and thus play an indispensable role in the hepatitis C virus replication.
  • ELAVL1 / HuR an intracellular molecule that specifically binds miRNA-122, which is known to bind, and revealed that ELAVL1 / HuR binds specifically and strongly to ligosertip.
  • the present invention was completed as a specific and effective inhibitor against HCV infection, and has a medicinal use as an anti-HCV therapeutic agent.
  • An object of the present invention is to provide a pharmaceutical composition that can prevent or treat liver disease caused by the new hepatitis C virus.
  • Another object of the present invention is to provide an antiviral composition for hepatitis C virus.
  • Another object of the present invention to provide a health functional food composition that can prevent or improve liver disease caused by hepatitis C virus.
  • Still another object of the present invention is to provide a method for preventing or treating liver disease caused by hepatitis C virus.
  • Still another object of the present invention is to provide a use for preventing or treating liver disease caused by hepatitis C virus.
  • Another object of the present invention is to provide an antiviral use against hepatitis C virus.
  • the present invention provides a novel use of ligosertip.
  • the present invention provides a use for the prevention or treatment of liver disease by hepatitis C virus of rigosertib or a pharmaceutically acceptable salt thereof.
  • the present invention provides an antiviral use against hepatitis C virus of rigosertib or a pharmaceutically acceptable salt thereof.
  • the present invention is a pharmaceutical composition for the treatment or prevention of hepatitis C virus (Hepatitis C virus, HCV) containing ligosertip (rigosertib) or a pharmaceutically acceptable salt thereof as an active ingredient To provide.
  • Hepatitis C virus Hepatitis C virus, HCV
  • ligosertip rigosertib
  • a pharmaceutically acceptable salt thereof as an active ingredient To provide.
  • the present invention also provides an antiviral composition against HCV, which contains ligosertip or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a nutraceutical composition for the improvement or prevention of liver disease by HCV containing the ligosertip or a food acceptable salt thereof as an active ingredient.
  • the present invention provides a method for preventing or treating liver disease caused by hepatitis C virus of rigosertib or a pharmaceutically acceptable salt thereof.
  • the present invention provides a novel use of antiviral agents against HCV and liver disease caused by HCV that can specifically and effectively inhibit HCV infection without hepatitis C virus treatment, prevention, antiviral use, and resistance problems.
  • Prophylactic or therapeutic compositions are provided.
  • Ligosertip according to the present invention can be a specific and effective treatment for HCV infection by inhibiting the binding of microRNA-122 and ELAVL1 / HuR. Moreover, unlike the anti-HCV treatments developed in the past, they do not target HCV proteins and inhibit the action of hepatocyte agonists in humans, which can solve the problem of resistance to the previously developed drugs. Combination therapy with known therapeutic agents is possible, and is also useful as a good treatment strategy for HCV that does not cause resistance.
  • 1 shows miRNA-122 directly binding to ELAVL1 / HuR and ELAVL1 / HuR efficacy verification results
  • 1A is the isomiR structure of miRNA-122
  • 1B is an RNA electro mobility shift assay (REMSA) using GST-HuR
  • 1C is the knockout result of intracellular molecule ELAVL1 / HuR using HCV subgenomic replicon capable of translation / replication assay
  • 1D is specifically knockout of Huh7 intracellular ELAVL1 / HuR using CRISPR / Cas9 system. This is the result of the Westing blot when you make it.
  • Figure 2A is a graph of the screening results of compounds inhibiting HCV translation / proliferation using the structure of subgenomic replicon (HIRL-2Aneo-NSrep) of HCV capable of reporter assay and Seleckchem cellular kinase inhibitor libray
  • 2B is a recombinant GST-HuR protein.
  • the result of direct binding between biotin-rigosertb and in vitro pulldown assay is shown.
  • 2C and 2D show the chemical structure of ligosertip and biotin-lygosertip
  • 2E shows the interaction between HCV and molecules in the cell. (ELAVL1 / HuR-microRNA-122-HCV translation / cloning) is shown.
  • Figure 3 is a graph re-validating the anti-HCV effect of ligosertip through HIRL-2Aneo-NSrep_FL Huh-7 luciferase assay.
  • treatment in the present invention means an approach for obtaining beneficial or desirable clinical results.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (which may be partial or total), detectable or not detected.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include the disorders to be prevented as well as the treatments required in the already occurring intestine. By “palliating" a disease, the extent to which the disease state and / or undesirable clinical signs and / or the time course of progression is slowed or lengthened, as compared to the case without treatment.
  • prevention in the present invention refers to a therapy that protects against the onset of a disease or disorder so that clinical symptoms of the disease do not develop.
  • prophylaxis refers to administering a therapy to a subject (eg, administering a therapeutic agent) before the indication of the disease is detectable in the subject (eg, to a detectable infectious agent (eg, To a subject in the absence of the virus).
  • a subject may be an individual at risk of developing a disease or disorder, such as an individual having one or more risk factors known to be associated with the development or onset of the disease or disorder.
  • the term “preventing HCV infection” refers to administering an anti-HCV therapeutic substance to a subject having no 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” refers to an amount effective to elicit a desired biological or medical response, such as an amount of a compound sufficient to effect such treatment for a disease when administered to a subject for treating the disease. Refers to.
  • 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 can include a range of amounts.
  • an effective amount may be one or more doses, ie a single dose or multiple doses may be required to achieve the desired treatment endpoint.
  • An effective amount can be considered in connection with administering one or more therapeutic agents, and a single agent can be considered to be provided in an effective amount if a desirable or beneficial result can be achieved or is achieved with one or more other agents.
  • Suitable doses of any co-administered compound can be optionally reduced due to the combined action of the compound (eg, additional or synergistic effects).
  • the present invention provides a method for preventing or treating liver disease caused by hepatitis C virus of rigosertib or a pharmaceutically acceptable salt thereof, and for use in a pharmaceutical composition for preventing or treating liver disease. It is about.
  • the present invention in one aspect, relates to antiviral use for hepatitis C virus, or use for antiviral composition against hepatitis C virus, of rigosertib or a pharmaceutically acceptable salt thereof.
  • the present invention provides a pharmaceutical composition for the treatment or prevention of hepatitis C virus (Hepatitis C virus, HCV) containing a rigosertib or a pharmaceutically acceptable salt thereof as an active ingredient. It relates to a composition.
  • Hepatitis C virus HCV
  • HCV hepatitis C virus
  • Rigosertipib (rigosertib) is a material known as an anticancer agent, specifically, a material having a structure as shown in the following formula (1).
  • Hepatitis C virus consists of a single stranded ⁇ 10-kb single stranded (+) sense RNA in molecular biology, belongs to flaviviridae, and utilizes an abnormal translation initiation process such as an internal ribosomal entry site. The translation process occurs and is cleaved by proteolytic enzymes encoded by intracellular proteases or viruses, respectively, to designate non-structural proteins that are important for the translation, replication, and proliferation of structural proteins or viruses, respectively. .
  • Existing studies have been shown to actively utilize intracellular molecules in the replication and translation process of HCV.
  • microRNA-122 microRNA-122 (miRNA-122, accounts for 70% of human hepatocyte microRNAs). Is known to play an essential role in the replication of hepatitis C virus.
  • ligosertip strongly inhibits replicon proliferation similarly to the danoprevir already developed by Roche and used in clinical trials, and shows an inhibitory effect of 80% or more even at low concentrations (0.2 ⁇ M).
  • the ligosertip of the present invention unlike the anti-HCV therapeutic agent developed in the past, inhibits the action of host factors (miRNA-122 and ELAVL1), which is not a protein produced by HCV itself (miRNA-122 and ELAVL1) / HuR), because it does not target the HCV protein, it can be used as a preventive or therapeutic agent for liver disease caused by the new concept hepatitis C virus, which can solve the resistance problems of previously developed drugs.
  • host factors miRNA-122 and ELAVL1
  • HuR HuR
  • the hepatitis C virus may be hepatitis C, hepatic fibrosis by hepatitis C virus, cirrhosis by hepatitis C virus, and liver cancer by hepatitis C virus.
  • the ligosertip may be characterized in that it is administered in combination with an HCV therapeutic agent, for example, sovaldi (sovaldi), Danoprevir (Daclatasvir).
  • HCV therapeutic agent for example, sovaldi (sovaldi), Danoprevir (Daclatasvir).
  • antiviral agents for example ribavirin.
  • the ligosertip may be used as an antiviral agent for hepatitis C virus, which inhibits the translation / replication / proliferation of hepatitis C virus, and thus, the present invention is in another aspect, the ligosertip or pharmaceutically acceptable thereof.
  • An antiviral composition for HCV containing possible salts as an active ingredient.
  • “Pharmaceutically acceptable salts” in the present invention refers to salts of any compound of the present invention that are not toxic or inappropriate for pharmaceutical use while maintaining the biological properties of the compound ligosertip of the present invention. Such salts can 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, glycolic acid, gluc Taric 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- (4-hydroxybenzoyl) benzoic acid, picric acid, cinnamic acid, mandelic acid, Phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzene sulfonic
  • salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium salts, and the like, if the compound contains basic functional groups, salts with non-toxic organic or inorganic acids, such as hydrohalides (eg hydro Chloride or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate Bate, Lactate, Malonate, Succinate, Sorbate, Ascorbate, Maleate, Maleate, Fumarate, Tartarate, Citrate, Benzoate, 3- (4-hydroxybenzoyl) benzoate, Peak Late, cinnamate, mandelate, phthalate, laurate, methanesulfonate mesylate), ethanesulfone , 1,2-e
  • the igorsertip or a pharmaceutically acceptable salt thereof is administered to a subject or patient, wherein the “subject (s)” are animals, eg non-primates (eg, cattle, pigs, horses, cats, Mammals, including, for example, dogs, rats and mice) and primates (eg, monkeys such as cynomolgous monkeys, chimpanzees and humans), and eg humans.
  • the subject does not resist or respond to current treatments for hepatitis C infection.
  • the subject is a livestock animal (eg, horse, cow, pig, etc.) or a pet animal (eg, dog or cat).
  • the subject is a human.
  • therapeutic agent (s) or therapeutic composition refers to any agent (s) that can be used to treat or prevent a disorder or one or more indications thereof.
  • therapeutic agent refers to a ligosertip, which is a compound of the present invention.
  • therapeutic agent does not refer to a compound of the present invention.
  • the therapeutic agent is an agent that is known to be useful for treating or preventing a disorder or one or more indications thereof, has been used for this purpose, or is currently in use.
  • the ligosertip used in the process of the invention preferably uses a compound of formula (I), where appropriate in the form of a salt, alone, or one or more suitable pharmaceutically acceptable carriers such as diluents or adjuvants, Or by using a pharmaceutical composition containing in form in combination with another anti-HCV agent.
  • the derivatives of the present invention can be administered by any conventional route, in particular by oral, parenteral, rectal route, or by inhalation (eg in aerosol form).
  • compositions for oral administration tablets, pills, hard gelatin capsules, powders or granules can be used.
  • the compounds according to the invention are admixed with at least one inert diluent or adjuvant such as sucrose, lactose or starch.
  • inert diluent or adjuvant such as sucrose, lactose or starch.
  • These compositions may comprise, in addition to diluents, substances such as, for example, lubricants such as magnesium stearate, or coatings for controlled release.
  • inert diluents such as pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing water or liquid paraffin can be used.
  • these compositions may also include, in addition to diluents, substances such as, for example, wetting agents, sweetening agents or flavoring agents.
  • the composition for parenteral administration may be an emulsion or a sterile solution.
  • solvents or vehicles propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil or injectable organic esters such as ethyl oleate can be used.
  • These compositions may also contain adjuvants, especially wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in a number of ways, for example by using a filter for bacteria, by irradiation or by heating. They can also be prepared in the form of sterile solid compositions that, when used, can be dissolved in sterile water or any other injectable sterile medium.
  • compositions for rectal administration are suppositories or capsules for rectal administration, which contain, in addition to the active ingredient, excipients such as cocoa butter, semisynthetic glycerides or polyethylene glycols.
  • the composition may also be an aerosol.
  • the composition When used in liquid aerosol form, the composition may be a stable sterile solution, or a solid composition which, when used, is dissolved in non-pyrogenic sterile water, saline or any other pharmaceutically acceptable vehicle.
  • the active ingredient 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, for example dextran, mannitol or lactose.
  • compositions of the invention are pharmaceutical compositions or single unit dosage forms.
  • Pharmaceutical compositions and single unit dosage forms of the invention may be used in a prophylactic or therapeutically effective amount of one or more prophylactic or therapeutic agents (eg, a compound of the invention, or other prophylactic or therapeutic agents), and typically one or more pharmaceutically acceptable Carriers or excipients.
  • prophylactic or therapeutic agents eg, a compound of the invention, or other prophylactic or therapeutic agents
  • Carriers or excipients typically one or more pharmaceutically acceptable Carriers or excipients.
  • the term "pharmaceutically acceptable” means that it can be used in animals, more specifically in humans, as listed in a pharmacopoeia that has been approved or generally accepted by the government or regulatory bodies of the government. do.
  • carrier refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete adjuvant)), excipient, or vehicle with which the therapeutic agent is administered.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils including oils or synthetic oils of petroleum, animal or vegetable origin, for example peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, in particular injectable solutions.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • compositions and dosage forms comprise one or more excipients.
  • Suitable excipients are known to those skilled in the pharmaceutical arts, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, flour, silica gel, sodium stearate, glycerol monostearate, Talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like.
  • suitable excipient to be incorporated into a pharmaceutical composition or dosage form depends on various factors known in the art, including the manner in which the dosage form is administered to the subject, the specific active ingredients included in the dosage form, and the like.
  • the composition or single unit dosage form may also contain small amounts of wetting or emulsifying agents, or pH buffers.
  • the invention also includes anhydrous pharmaceutical compositions and dosage forms comprising the active ingredient.
  • water eg 5%
  • the addition of water is widely accepted in the pharmaceutical industry as a simulated long term storage means for determining the properties (eg shelf life or stability) of a formulation over time.
  • water and heat accelerate the decomposition of some compounds.
  • water and / or moisture typically occur during the manufacture, handling, packaging, storage, transportation and use of the formulation, so the effect of water on the formulation can be very important.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture components and low moisture or low humidity conditions. If it is anticipated to be in substantial contact with moisture and / or moisture during manufacture, packaging and / or storage, pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising primary or secondary amines may be in anhydrous form. Is preferably.
  • Anhydrous pharmaceutical compositions should be prepared and stored so that their anhydrous properties are maintained. Therefore, it is desirable to package using known materials to prevent the anhydrous composition from exposure to water and to include it in a suitable specification kit. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.
  • compositions and dosage forms comprising one or more compounds that reduce the rate of degradation of the active ingredient.
  • compounds include, but are not limited to, antioxidants (eg, ascorbic acid), pH buffers, or salt buffers.
  • compositions and single unit dosage forms can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations 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 contain a prophylactic or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in purified form, together with a suitable amount of carrier, and will be provided in a form suitable for administration to a subject.
  • the formulation should be suitable for the mode of administration.
  • 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, and most preferably a human subject.
  • compositions of the invention are formulated to be suitable for their intended route of administration.
  • routes of administration include parenteral routes of administration such as intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, oral, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, synovial and There is, but is not limited to, a rectal route of administration.
  • the composition is formulated into a pharmaceutical composition that has been modified for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to a human according to conventional procedures.
  • the pharmaceutical composition is formulated into a composition that is administered subcutaneously to a human according to conventional procedures.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include solubilizers and local anesthetics (eg lignocaine) to soothe pain at the injection site.
  • dosage forms include tablets; Caplets; Capsules such as soft elastic gelatin capsules; Casein; Troches; Lozenges; Dispersion liquids; Suppositories; Ointment; Poultice (foam medicine); Paste; Powder; dressing; cream; salve; Solution; patch; Aerosols (eg, nasal sprays or inhalers); Gels; Liquid dosage forms suitable for oral or mucosal administration to a subject, including suspensions (eg, aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions and elixirs; Liquid dosage forms suitable for parenteral administration to a subject; And sterile solids (eg, crystalline or amorphous solids) which, when restored, can provide a liquid dosage form suitable for parenteral administration to a subject.
  • suspensions eg, aqueous or non-aqueous liquid suspensions, oil-in-water
  • compositions, forms and dosage forms of the invention will typically vary depending on their use.
  • the dosage form used for the initial treatment of a viral infection may contain one or more active ingredients in an amount greater than the amount included in the dosage form used for the maintenance therapy of the viral infection.
  • parenteral dosage forms may contain one or more active ingredients in an amount less than that included in oral dosage forms used to treat the same disease or disorder.
  • Other ways of modifying this mode and the specific dosage forms encompassed by this invention will already be apparent to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • compositions of the present invention are generally anhydrous lyophilized powders or water-free concentrates in sealed containers, such as ampoules or sachets, for example indicative of the amount of active agent, mixed separately or together in a unit dosage form. It is provided in the form.
  • the composition When the composition is administered by infusion, the composition may be dispensed into an infusion container containing sterile pharmaceutical grade water or saline.
  • sterile water or saline ampoules for injection may be provided to allow the components to be mixed prior to administration.
  • Conventional dosage forms of the present invention comprise a compound of the present invention, ligosertip, or a pharmaceutically acceptable salt, solvate or hydrate thereof in an amount ranging from about 0.1 mg to about 1000 mg / day, which is in the morning A single dose once a day or divided doses with meals preferably for one day.
  • compositions of the invention suitable for oral administration may be in separate dosage forms, such as tablets (eg chewing tablets), caplets, capsules and liquids (eg flavor syrups) and the like.
  • dosage forms contain a predetermined amount of active ingredient and can be prepared by methods of pharmacy known to those skilled in the art. In general, see Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • oral dosage forms are solid and are prepared under anhydrous conditions using anhydrous ingredients as detailed in the paragraphs above.
  • compositions of the invention are prepared by combining the active ingredient (s) as an intimate mixture with one or more excipients according to conventional pharmaceutical synthesis techniques.
  • Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • excipients suitable for use in liquid or aerosol dosage forms for oral administration 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 include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants Does not.
  • solid excipients When solid excipients are used, they appear in the most advantageous oral dosage unit form because tablets and capsules 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 of the methods of preparation.
  • compositions and dosage forms are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then molding the product, if necessary, into the desired shape.
  • tablets can be made by compression or molding.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free flowing form (eg, powder or granules), optionally mixed with excipients.
  • Molded tablets may be made by molding in a suitable machine a mixture of powdered compounds moistened with an inert liquid diluent.
  • excipients that can be used in oral dosage forms of the 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, powder tragacanth, guar gum, Cellulose and its derivatives (e.g. ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, (e.g. , 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
  • natural and synthetic gums such as acacia gum, sodium alginate, alginic acid, other alginates, powder tragacanth, guar gum,
  • fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include talc, calcium carbonate (eg, granules or powders), microcrystalline cellulose, powdered cellulose, dexrate, kaolin, mannitol, silicic acid, sorbitol, starch , Pregelatinized starch, and mixtures thereof, but is not limited thereto.
  • the binder or filler included in the pharmaceutical composition of the present invention is typically present in an amount of about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
  • Suitable forms of microcrystalline cellulose include AVISEL PH 101, AVICEL PH 103, AVICEL RC 581, AVICE PH 105 and are commercially available (FMC Corporation, American Viscose Division, Avicel Sales). Sales; commercially available from Markus Hook, Pennsylvania, USA), and mixtures thereof.
  • disintegrants in the compositions of the present invention provides tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate upon storage, and tablets containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, a sufficient amount of disintegrant should not be used to form the solid oral dosage form of the present invention, which is neither too much nor too little to deleteriously alter the release of the active ingredient.
  • the amount of disintegrant used depends on the type of formulation and can be readily determined by one skilled in the art. Typical pharmaceutical compositions comprise about 0.5 to about 15 weight percent, specifically about 1 to about 5 weight percent disintegrant.
  • Disintegrants that can be used in the pharmaceutical compositions and dosage forms of the invention include agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polyacrylic potassium, sodium starch glycolate, potato or tapioca starch, Pregelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
  • Lubricants that can be used in the pharmaceutical compositions and dosage forms of the present invention include calcium stearate, magnesium stearate, mineral oil, mineral diesel, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate Talc, hydrogenated vegetable oils (e.g. peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, and their Mixtures are but are not limited to these.
  • hydrogenated vegetable oils e.g. peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, corn oil and soybean oil
  • zinc stearate ethyl oleate, ethyl laurate, agar, and their Mixtures are but are not limited to these.
  • Additional lubricants include, for example, siloid silica gel (AEROSIL 200, manufactured by WR Grace Co., Baltimore, MD), solidified aerosol of synthetic silica (Degussa Corporation) (Available from Degussa Co., Piano, Tex.), CAB O SIL (pyrogenic silicon dioxide product, from Cabot Co .; Boston, Mass.), And mixtures thereof. .
  • AEROSIL 200 siloid silica gel
  • Degussa Corporation solidified aerosol of synthetic silica
  • CAB O SIL pyrogenic silicon dioxide product, from Cabot Co .; Boston, Mass.
  • lubricants are typically used in amounts less than about 1% by weight of the pharmaceutical composition or dosage form into which they are incorporated.
  • the ligosertip of the present invention can be administered, for example, by controlled release means or delivery devices known to those skilled in the art.
  • dosage forms can be, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, particulates, liposomes, microspheres, or combinations thereof to provide a desired release profile at various ratios.
  • Water may be used to delay or release 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 includes single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps and caplets, etc., modified to controlled release.
  • controlled release drugs have a common purpose to provide improved drug therapies than are achieved by their corresponding non-controlled release drugs.
  • the use of controlled release formulations that are best designed for medical treatment is characterized by the use of a minimum 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, a decrease in the number of doses and an increase in compliance of the subject.
  • controlled release formulations may be used to affect the time of onset of the drug or other properties such as blood levels of the drug, thus affecting the development of side effects (adverse effects).
  • Controlled release of the active ingredient can be stimulated by a variety of conditions including pH, temperature, enzymes, water, or other physiological conditions or compounds, and the like.
  • the drug can be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration.
  • a pump can be used (Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987)); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989).
  • polymeric materials can be used.
  • a controlled release system can be located at a suitable site determined by one of skill in the art in a subject, ie only a portion of the systemic dosage is required (eg, 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 is a solid internal matrix such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene , Polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate, copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrogels of hydrophilic polymers such as acrylic acid and methacrylic acid, collagen, It can be dispersed in crosslinked polyvinylalcohol and partially hydrolyzed crosslinked polyvinyl acetate, which is an external polymer membrane such as polyethylene, polypropylene, ethylene / propylene copolymer, ethylene / ethyl acrylate copolymer, ethylene / vinyl Acetate copolymer, silicone rubber, polydimethyl siloxane, four Friendly rubber, chlorinated polyethylene, polyvinyl
  • parenteral dosage forms can be administered to a subject by a variety of routes including subcutaneous, intravenous (including bolus injection), intramuscular and intraarterial routes of administration, and the like. Because these parenteral routes of administration typically bypass the subject's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized prior to administration to the subject. Examples of parenteral dosage forms include, but are not limited to, injectable solutions, anhydrous products that can be dissolved or suspended in pharmaceutically acceptable injectable vehicles, injectable suspensions, and emulsions.
  • Suitable vehicles are known to those skilled in the art that can be used to provide the parenteral dosage forms of the invention. Examples of these include water for injection (USP); Aqueous vehicles such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, and the like; Water miscible vehicles such as ethyl alcohol, polyethylene glycol, polypropylene glycol, and the like; And non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate, and the like. In addition, compounds that increase the solubility of one or more active ingredients disclosed herein may also be incorporated into the parenteral dosage forms of the invention.
  • Transdermal, topical and mucosal dosage forms of the 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, eg, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000) and Introduction to Pharmaceutical dose form, 4th ed., Lea & Febiger, Philadelphia (1985). )].
  • Suitable dosage forms for treating oral mucosal tissue may be formulated as oral cleaning solutions or oral gel formulations.
  • transdermal dosage forms include "reservoir” or “matrix” patches, which may be skin or wound The site may be applied for a certain period of time to infiltrate the desired amount of active ingredient.
  • Suitable excipients included in the present invention, and other materials that can be used to provide transdermal, topical and mucosal dosage forms, are known to those of ordinary skill in the art of pharmacy, and they are given in any given pharmaceutical composition or dosage form. It depends on the specific organization to which it applies. In view of this fact, conventional excipients include water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl, forming lotions, tinctures, creams, emulsions, gels or ointments.
  • conventional excipients include water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl, forming lotions, tinctures, creams, emulsions, gels or ointments.
  • humectants or humectants may also be added to the pharmaceutical compositions and dosage forms. Examples of such additional ingredients are known in the art. See, eg, Remington's Pharmaceutical Sciences, 16th, 18th and 20th eds., Mack Publishing, Easton PA (1980, 1990 & 2000).
  • additional ingredients may be used prior to treatment with the active ingredients of the invention, used with the active ingredients of the invention, or after treatment with the active ingredients of the invention.
  • 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; Dimethyl acetamide; Dimethyl formamide; Polyethylene glycol; Pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (povidone, polyvidone); Urea; And various water soluble or water insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).
  • 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 delivery of one or more active ingredients.
  • the delivery can be improved by adjusting the polarity of the solvent carrier, its ionic strength or tension.
  • the delivery may also be improved by adding a compound, such as stearate, to the pharmaceutical composition or dosage form to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients.
  • stearates may act as liquid vehicles for formulation, emulsifiers or surfactants, and delivery or penetration enhancers.
  • Other salts, hydrates or solvates of the active ingredient can be used to further adjust the properties of the resulting composition.
  • the physician When treating a human, the physician will determine the dosage that is considered most appropriate, depending on the prophylactic or curative treatment, and the age, weight, stage of infection and other factors specific to the subject to be treated. Generally, for adults, 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 about 5 to about 400 mg / day, more preferably 25 to 200 mg / day, per adult. Also preferred is a dosage rate of about 50 to about 500 mg / day.
  • the present invention provides a method of treating or preventing a hepatitis C virus infection by administering to a subject in need thereof an effective amount of a compound of the invention having a high therapeutic index for the hepatitis C virus or a pharmaceutically acceptable salt thereof.
  • Hepatitis C virus infection a method for treating or preventing liver disease caused by such a virus.
  • the therapeutic index can be measured according to any method known to those skilled in the art, such as those described in the Examples below.
  • the therapeutic index is the ratio of the concentration of the compound that is toxic to the concentration of the compound effective for hepatitis C virus.
  • Toxicity can be measured by any technique known to those of skill in the art, including cytotoxicity (eg IC50 or IC90) and lethal dose (eg LD50 or LD90).
  • the effective concentration can be measured using any technique known to those skilled in the art, including effective concentrations (eg, EC50 or EC90) and effective doses (eg, ED50 or ED90).
  • similar measurements are compared in proportions (eg IC50 / EC50, IC90 / EC90, LD50 / ED50 or LD90 / ED90).
  • 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 present invention effective to prevent, treat, manage or ameliorate a disorder or one or more indications thereof will vary depending on the nature and severity of the disease or condition and the route by which the active ingredient is administered.
  • the frequency and dosage of administration may also be determined for each subject depending on the particular therapy (eg, therapeutic or prophylactic) administered, the severity of the disorder, disease or condition, the route of administration as well as the subject's age, weight, response and past history. It will depend on the specific element. Effective dosages may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • composition dosages are amounts of active compound (mg or ⁇ g) per kilogram of subject or sample, for example about 10 ⁇ g / kg to about 50 mg / kg, about 100 ⁇ g / kg to about 25 mg / kg, or about 100 ⁇ g / kg to about 10 mg / kg.
  • the dosage administered to a subject is typically 0.140 mg to 3 mg per kg body weight of the subject based on the weight of the active compound.
  • the dosage administered to a subject is 0.20 mg to 2.00 mg or 0.30 mg to 1.50 mg per kg body weight of the subject.
  • the daily dosage range of the compositions of the present invention proposed for the symptoms described herein is about 0.1 to about 1000 mg / day, which is a single dose once a day or divided doses throughout the day. In one embodiment, the daily dose is divided into equal amounts and administered twice daily.
  • the daily dosage range should be about 10 to about 200 mg / day, more specifically about 10 to about 150 mg / day, even more specifically about 25 to about 100 mg / day.
  • the clinician or therapist will know when and how to stop, adjust or terminate the therapy with respect to the subject's response.
  • compositions of the present invention can be administered for different diseases and conditions.
  • an amount sufficient to prevent, manage, treat or ameliorate the disorder but not sufficient to cause side effects associated with the compositions of the present invention or sufficient to reduce the side effects is also included in the dosage and frequency schedule described above. do.
  • 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 ligosertip of the present invention or a food acceptable salt thereof may be used for the improvement or prevention of liver disease caused by hepatitis C virus, and thus the present invention provides the ligosertip or a foodstuff thereof.
  • the present invention relates to a nutraceutical 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 prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and improving liver disease caused by HCV.
  • health functional food used in the present invention refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and the structure and function of the human body. It means the ingestion for the purpose of obtaining a useful effect for health use such as nutrient control or physiological action.
  • the health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards.
  • Food Additive Reduction examples include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high amount pigment and guar gum; And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
  • the health functional food in the form of tablets is granulated in a conventional manner by mixing a mixture of ligosertip, an active ingredient of the present invention with excipients, binders, disintegrants and other additives, and then compressed with a lubricant and the like. Or the mixture can be directly compression molded.
  • the health functional food in the form of tablets may contain a mating agent or the like as necessary.
  • Hard capsules of the health functional food in the form of capsules can be prepared by filling a conventional hard capsules mixture of a mixture of ligosertip, which is the active ingredient of the present invention, with additives such as excipients, and the soft capsule is excipient
  • the mixture mixed with such additives can be prepared by filling in a capsule base such as gelatin.
  • the soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
  • the health functional food in the form of a cyclic form may be prepared by molding a mixture of the compound of Formula 1, an excipient, a binder, a disintegrant, etc., which is the active ingredient of the present invention by a known method, and if necessary, sucrose or other It may be encapsulated with a skin coating, or the surface may be coated with a material such as starch, talc.
  • the health functional food in the form of granules can be prepared in the form of a mixture of a mixture of ligosertip and excipients, excipients, binders, disintegrants, etc., which are the active ingredients of the present invention, by a conventionally known method. And the like.
  • the health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.
  • 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.), Were searched. It was analyzed by intracellular molecules that bind to biotinylated miRNA-122 using LC-MS / MS technique. As a result, ELAVL1 / HuR was identified and specific binding between miRNA-122 and ELAVL1 / HuR was removed. In order to monitor transcription / cloning of HCV at knockout), the following experiment was performed.
  • FIG. 1A There are four types of isomiR for miRNA-122 in liver cells (FIG. 1A). Specific binding between miRNA-122s was confirmed by RNA electro-mobility shift assay (REMSA), and specific recombinant ELAVL1 / HuR purification was performed. And REMSA process is as follows.
  • Esherichia coli Recombinant glutathione from plasmid pGEX-KG-HuR (WT) using strain BL21 (DE3) pLysS (Promega) S ELAVL1 / HuR (GST-HuR) fused to transferase (GST) was produced.
  • Isopropyl- ⁇ -D-thio galactopyranoside was added to induce GST-HuR protein expression at OD 600 0.5.
  • cells are harvested and lysed [20 mM phosphate (pH 7.6), 300 mM NaCl, 0.5 mM phenyl methane sulfonylfluoride (PMSF), 1 mM ⁇ -mercaptoethanol and 10% glycerol (v / v)] followed by sonication.
  • the resulting cell extracts were loaded into glutathione agarose 4B (Peptron, Dajeon, Korea), washed with lysis buffer, and bound GST-HuR was eluted with reduced glutathione.
  • RNA-protein complex was added to a 5% non-denatured gel. Analyzed by auto radiography.
  • the wild type had only one nucleotide difference in the 3 ′ end region of miRNA-122, the binding of which was the strongest, indicating that the 3 ′ end region of miRNA-122 was very important for binding to ELAVL1 / HuR.
  • 1B shows that ELAVL1 / HuR accounts for 70% of human hepatocyte miRNA and binds directly to miRNA-122, which is known to play an essential role in the replication process of hepatitis C virus.
  • luciferase-expressing hepatitis C subgenomic replicons derived from the infectious hepatitis C virus production system (pJFH1) were introduced and identified using the CRISPR / Cas9 system ELAVL1 / HuR.
  • pJFH1 infectious hepatitis C virus production system
  • Knockout using the CRISPR / Cas9 system of the target genes was performed using pLentiCRISPRv2 (Addgene) and stably transduced Huh7 cells were selected using puromycin. Cells were selected with puromycin and collected and colonies of selected Huh7 cells were analyzed using Western blot 30 days after infection. Genomic DNA of selected colonies was isolated using the Genomic DNA Column Kit (Zymo Reserch), amplified from genomic DNA adjacent to the ELAVL1 / HuR target site for the inserted guide RNA sequence and DNA sequencing of the resulting amplicon sequences (Cosmo). Genentech).
  • pSGR-JFG1 / Luc-GND (Ralf Bartenschlager), which is a mutant of the activation site of pSGR-JFG1 / Luc or NS5B, which is a subgenomic replicon capable of two types of Rutherase assays, is digested with restriction enzymes and then in vitro transcription is performed.
  • RNA of luciferase-expressing hepatitis C subgenomic replicon was synthesized.
  • the naive Huh7 cell line or CRISPR / Cas9 gene shears were used to transduce ELAVL1 / HuR knockout cells using electroporation (1 ug of RNA into 1 ⁇ 10 ⁇ 6 cells).
  • Fractionated cells were lysed with 1 ⁇ passive lysis buffer (Promega), and a portion of the lysate was analyzed by the Dual-Luciferase® Reporter Assay System (Promega), and the firefly luciferase signal was normalized to Renilla signal or total protein amount.
  • HCV subgenomic replicons are available. Primary screening was attempted using inhibitor libraries for various kinases in the cell.
  • the Huh7 cell line containing assayable HCV 1b subgenomic replicon HIRL-2Aneo-NSrep was plated, followed by treatment with Selleckchem cellular kinase inhibitory chemicals (Korea Research Institute of Bioscience and Biotechnology) to a concentration of 1 ⁇ M, followed by luciferase assay 24 hours later was performed.
  • Fractionated cells were lysed with 1 ⁇ passive lysis buffer (Promega), and a portion of the lysate was analyzed by the Dual-Luciferase® Reporter Assay System (Promega), and the firefly luciferase signal was normalized to Renilla signal or total protein amount.
  • Example 2 In order to reverify that the ligosertip identified in Example 2 is a specific and effective inhibitor against HCV infection, the following experiment was additionally performed.
  • HCV 1b subgenomic replicon HIRL-2Aneo-NSrep and firefly luciferase were assayed for HCV-specific inhibitor ligosertip, which was discovered using a subgenomic replicon of HCV capable of reporter assay in the same line as the screening result of Example 2.
  • Huh7 cell line was plated by 2 ⁇ 10 ⁇ 4 cells in 24 wells, 1 ⁇ M of rigosertib was treated for 0, 24, 48, and 72 hours, and then fractionated at each time period to perform luciferase assay.
  • 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). Renilla signals were normalized to firefly luciferase signals.
  • the inhibition mechanism of ligosertip according to the present invention unlike the anti-HCV therapeutic agent developed in the past, inhibits the action of host factors (acting molecules of human hepatocytes) that are not proteins produced by HCV itself (miRNA- Because it does not target 122 and ELAVL1 / HuR) and HCV proteins, it is expected to be a new concept of anti-hepatitis C virus that can solve the problem of resistance to drug. It is expected to be a good treatment strategy for HCV which is inexpensive and does not cause resistance because it is used alone or in combination with previously known ribavirin.
  • SEQ ID NO: 1 Wild type of miR-122
  • SEQ ID NO: 2 (Isomir of miR-122 (+ A)):
  • SEQ ID NO: 3 (Isomir of miR-122 (+ U)):
  • SEQ ID NO: 4 (Isomir of miR-122 (-G)):

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Abstract

La présente invention concerne la nouvelle utilisation du rigosertib et, plus particulièrement, l'utilisation du rigosertib, qui peut inhiber spécifiquement et efficacement une infection par le VHC sans problème de résistance, dans la prévention ou le traitement d'une maladie du foie causée par le virus de l'hépatite C et dans l'activité antivirale du virus de l'hépatite C. En inhibant la liaison de microARN-122 à ELAVL1/HuR, le rigosertib selon la présente invention peut être un agent thérapeutique spécifique et efficace pour une infection par le VHC. En outre, contrairement aux agents thérapeutiques anti-VHC préexistants développés, le rigosertib inhibe l'activité de molécules agissant dans les hépatocytes humains sans cibler une protéine du VHC et peut ainsi résoudre le problème de résistance aux medicaments posé par les médicaments développés préexistants. Par conséquent, le rigosertib peut être utilisé seul ou en association avec des agents thérapeutiques déjà connus et avoir des applications utiles dans une bonne stratégie thérapeutique de traitement de VHC sans causer une résistance.
PCT/KR2018/002938 2017-03-14 2018-03-13 Nouvelle utilisation du rigosertib pour le traitement d'une maladie causée par le virus de l'hépatite c Ceased WO2018169283A1 (fr)

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