WO2014146218A1 - 马替麦考酚酯或其盐类用于制备抗流感病毒之药物的用途 - Google Patents

马替麦考酚酯或其盐类用于制备抗流感病毒之药物的用途 Download PDF

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WO2014146218A1
WO2014146218A1 PCT/CN2013/000336 CN2013000336W WO2014146218A1 WO 2014146218 A1 WO2014146218 A1 WO 2014146218A1 CN 2013000336 W CN2013000336 W CN 2013000336W WO 2014146218 A1 WO2014146218 A1 WO 2014146218A1
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influenza
influenza virus
virus
acceptable salt
drug
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PCT/CN2013/000336
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English (en)
French (fr)
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李安荣
朱纪洪
张温良
姚振文
黄文鑫
鲍力恒
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当代绿能科技股份有限公司
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Priority to CN201380074926.9A priority Critical patent/CN105246484B/zh
Priority to PCT/CN2013/000336 priority patent/WO2014146218A1/zh
Priority to US14/779,279 priority patent/US10221152B2/en
Publication of WO2014146218A1 publication Critical patent/WO2014146218A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/87—Benzo [c] furans; Hydrogenated benzo [c] furans
    • C07D307/88—Benzo [c] furans; Hydrogenated benzo [c] furans with one oxygen atom directly attached in position 1 or 3
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to the use of Mycophenolate Mofetil or a pharmacologically acceptable salt thereof, etc., for the preparation of a medicament for combating influenza virus. Background technique
  • Influenza virus (referred to as influenza virus) is one of the most threatening viruses in human life.
  • Four influenza pandemics occurred in the 20th century, from 1918 to 1919 (Spanish flu, H1N1), 1957 (Asian flu, H2N2), 1968 (Hong Kong flu, H3N2) and 1977 (Russian flu, H1N1).
  • the Spanish flu was the most serious in 1918, and its H1N1 strain was caused by avian influenza and human influenza viruses, causing 20 to 40 million deaths worldwide.
  • Mexico launched a new H1N1 flu epidemic, and more than one million people were suspected to have been infected, causing more than 10,000 deaths. It is also spreading ( Neumann, G.; Noda, T.; Kawaoka, Y.
  • Influenza virus is a negative-chain single-strand RA virus that causes influenza in humans and animals. It belongs to the Orthomyxoviridae family. It can be divided into A according to the characteristics of viral nucleoprotein, genetic material and matrix protein antigen. , B, C three serotypes. Among them, influenza A virus can cause influenza between different hosts, because the two antigenic hemagglutinin (HA) and neuraminidase, NA of the virus are multi-type, 16 kinds have been found before.
  • HA hemagglutinin
  • NA of the virus are multi-type, 16 kinds have been found before.
  • HA Hl ⁇ 16
  • 9 kinds of NA N1 ⁇ N9
  • B-type influenza virus antigen mutations rarely cause regional infection
  • type C influenza virus mainly uses pig as host
  • Human infections are rare, so the above-mentioned worldwide pandemic is an epidemic caused by influenza A virus.
  • influenza A virus can be divided into three parts: outer membrane, matrix protein and core.
  • the outer membrane has about 500 radially outwardly protruding protrusions, which are the two antigen types: columnar protrusion (HA) and mushroom-like protrusion (NA).
  • HA can agglutinate with surface receptors of various animal red blood cells, and can be divided into heavy chain and light chain after cleavage, so that the virus and host cells can fuse with each other; NA mainly has the activity of hydrolyzing sialic acid, cutting off the virus and the host cell.
  • the connection causes the virus to fall off from the adsorbed red blood cells, prevent the virus from accumulating, and promote the movement in the mucus.
  • the matrix protein is composed of Ml and M2, and has a knot that protects the virus core and maintains the virus. Structure.
  • the core is composed of 8 negative-stranded single-stranded RNA fragments that are entangled with ribonucleoprotein (NP) and RNA polymerase (PB1, PB2 and PA) to form ribonucleoprotein bodies.
  • NP ribonucleoprotein
  • PB1, PB2 and PA RNA polymerase
  • the reason why the flu virus causes panic is mainly because its variants will produce new subtypes and we are caught off guard.
  • antigenic drift which refers to influenza virus.
  • the point mutation of the type of antigen ( ⁇ ) amino acid produces small mutations; the other is the antigenic shift, which is the most common type of influenza virus mutation, and an antigenic mutation occurs every ten years.
  • influenza occurs in autumn and winter and early spring.
  • the target of invasion is respiratory mucosal epithelial cells, which multiply in the host cells, leading to mucosal congestion, edema, and cell degeneration and shedding.
  • the incubation period usually ranges from 1 to 3 days. It begins to develop symptoms such as fever, chills, headache, stuffy nose, and body aches. When it spreads to the lower respiratory tract, it may cause bronchitis and interstitial pneumonia. Because the influenza virus reduces respiratory mucosal epithelial cells.
  • M2 protein inhibitor acts on the viral transmembrane protein M2 ion channel, hindering H+ entry into the virus.
  • M2 protein inhibitor acts on the viral transmembrane protein M2 ion channel, hindering H+ entry into the virus.
  • These drugs are adamantane derivatives such as amantadine and rimantadine;
  • the two are neuraminidase inhibitors, which act on neuraminidase, which prevents the virus from hydrolyzing sialic acid and thus cannot leave the host cell and prevent the spread of the virus.
  • RNA Inhibitor of RNA polymerase which mainly inhibits the pathway of RNA polymerase (PB1, PB2, PA) synthesis of viral proteins, the drug has 2'-de -2'-deoxy-2,-fluoroguanosine (FdG), T-705; treatments such as interferon and siRNA (small interfering RNAs) are also used to prevent viral infection (Clercq, ED Antiviral) Agents Active against Influenza A Viruses. Nat. Rev. Drug. Discov. 5: 1015-1025, 2006).
  • influenza A virus has large antigenic variability and is highly contagious. Every year, about 500 million people in the world are infected with the flu, which often causes high mortality. It has a huge burden on the society and economic losses. Therefore, it is actively developing new anti-influenza drugs. It is an important issue. Summary of the invention
  • the present invention is the first to propose that Mycophenolate Mofetil or a salt thereof has an anti-influenza virus effect.
  • the present invention provides the use of martin's mycophenolate mofetil or a pharmacologically acceptable salt thereof, etc., for the manufacture of a medicament for use in combating influenza virus.
  • the pharmaceutically acceptable salt or the like may be a hydrochloride, a bromate or an organic acid salt.
  • the medicament of the present invention can be used in combination with one or more anti-influenza agents, wherein the agent is selected from the group consisting of an M2 inhibitor, an NA inhibitor, a R A polymerase inhibitor, an interferon, and a SiRNA.
  • the agent is selected from the group consisting of an M2 inhibitor, an NA inhibitor, a R A polymerase inhibitor, an interferon, and a SiRNA.
  • the agent can be (3R, 4R, 5S)-4-acetamide-5-amino-3-(1-ethylpropoxy)-1-cyclohexane) 1-carboxylic acid (oseltamivir, oseltamivir phosphate; flu, Tamiflu).
  • the present invention unexpectedly finds that the malteticin or a salt thereof has an efficacy of inhibiting the influenza phenotype of the drug resistant phenotype, and therefore, the present invention provides a martinisol or a pharmacologically acceptable thereof Use of acceptable salts in the manufacture of a medicament for combating a phenotype of influenza virus.
  • the influenza virus of the drug-resistant phenotype may be a H1N1 or H3N2 resistant strain, and in a preferred embodiment of the invention, the drug-resistant influenza virus is an influenza-resistant influenza virus strain.
  • the invention also provides a method of treating an influenza disease in a subject comprising administering an effective amount of malitol mycophenolate or a pharmacologically acceptable salt thereof to an individual in need thereof.
  • the method of the invention is to administer an effective amount of the drug to an individual infected with H1N1, H3N2 or a resistant strain thereof.
  • the method of the invention is to administer an effective amount of the drug to an individual infected with an influenza virus strain that is resistant to influenza.
  • matte mycophenolate or a pharmacologically acceptable salt thereof may be used in combination with one or more other anti-influenza agents.
  • Figure 1 is a graph showing the results of fluorescent immunostaining for detecting the efficacy of different concentrations of drugs against drug-resistant influenza virus strains by fluorescent immunostaining;
  • Fig. 2 is a graph showing the results of analyzing the efficacy of each compound against influenza virus in a mouse animal model, wherein (A) shows the survival rate of mice under different conditions; (B) is a graph of changes in body weight of mice. detailed description
  • ⁇ refers to the quantity of at least one (one or more).
  • the "influenza virus” described in this article is a cartridge for the influenza virus. It is a negative-chain single-strand RA virus that causes influenza in humans and animals. It belongs to the Orthomyxoviridae family. The characteristics of viral nucleoprotein, genetic material and matrix protein antigen can be divided into influenza A virus, influenza B virus and influenza C virus, and then according to the antigenicity of hemagglutinin and neuraminidase. type. According to the World Health Organization (WHO) influenza virus strain, the name contains six elements: type 'host/separation area/strain number/year of separation (HnNn), where for human influenza virus, host information is omitted, for type B and C The influenza virus omits subtype information.
  • WHO World Health Organization
  • influenza viruses referred to in the present invention include influenza A virus, influenza B virus, and influenza C virus.
  • influenza virus is, in particular, H1N1, H3N2 or a resistant strain thereof, particularly a strain of a strain resistant to Tamiflu.
  • Mycophenolate Mofetil or a pharmaceutically acceptable salt thereof and the like have an effect against influenza virus, thereby providing Mycophenolate Mofetil or its pharmacology Use of an acceptable salt for the preparation of a medicament for use against influenza viruses.
  • Mycophenolate Mofetil means 2-morpholinylethyl (E)-6-(l,3-dihydro-4-hydroxy-6-methoxy-7 - mercapto-3-oxo-5-isobenzofuran) -4-methyl-4-hexenoic acid ethyl ester (2-morpholinoethyl(E)-6-(l,3-dihydro-4-hydroxy- 6-methoxy-7-methyl-3-oxo-5-isobenzo iuranyl)-4-methyl-4-hexenoate), having the following structural formula:
  • salts refers to salts which are safe and effective for administration to humans or mammals and which have the desired biological activity.
  • Pharmaceutically acceptable salts include, but are not limited to, acidic or basic salts of the present invention, such as hydrochloric acid, hydrobromic acid, iodic acid, nitric acid, sulfuric acid, sodium hydrogen sulfate.
  • An alkaline salt synthesized by methanesulfonic acid or p-toluenesulfonic acid or an alkaline salt synthesized with aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine salts.
  • the pharmaceutically acceptable salt can be the hydrochloride salt.
  • the medicament can be used in combination with one or more anti-influenza agents, including but not limited to M2 protein inhibitors, such as adamantane derivatives of amantadine and rimantadine. ); Neuraminidase (NA) inhibitors, such as oseltamivir (influenza), zanamivir (zanamivir), peramivir, and cyclopentane Or 17 pyrrolidine derivatives; RNA polymerase inhibitors, such as 2'-deoxy-2'-fluoroguanosine (FdG), T- 705; and interferon or siRNA, etc.
  • M2 protein inhibitors such as adamantane derivatives of amantadine and rimantadine.
  • NA Neuraminidase
  • oseltamivir influenza
  • zanamivir zanamivir
  • peramivir peramivir
  • RNA polymerase inhibitors such as
  • the anti-influenza virus agent is Tamiflu, namely (3R, 4R, 5S)-4-acetamide-5-amino-3-(1-B Propyloxy)-1-cyclohexanyl)-1-carboxylic acid ((3 R,4 , 5 S)-4-acetamido-5 -amino-3 -(1 -ethy lpropoxy )-1 -cy clohexane- 1 - carboxylic acid; oseltamivir phosphate).
  • the medicament may include, but is not limited to, parenteral or oral administration.
  • the parenterally administered drug may be in the form of a solution, a suspension, an emulsion, and a solid injectable composition which can be dissolved or suspended in a solvent before use.
  • the injection can be prepared by dissolving, suspending or emulsifying one or more active ingredients in a diluent. Examples of the aforementioned diluent are distilled water for injection, physiological saline, vegetable oil, alcohols, and combinations thereof. Further, the injection may contain a stabilizer, a solubilizer, a suspending agent, an emulsifier, a smoothing agent, a buffer, a preservative, and the like. The injections are sterilized in the final formulation step or prepared in a sterile procedure.
  • the medicament of the present invention may also be formulated as a sterile solid formulation, for example, by lyophilization, and may be sterilized or dissolved in sterile injectable water or other sterile diluent prior to use.
  • the drug can also be administered orally, wherein the composition 13 000336
  • Solid compositions include tablets, pills, capsules, dispersible powders, granules and the like. Oral compositions also include mouthwashes and sublingual tablets.
  • Plastic bottles include hard plastic bottles and soft plastic bottles.
  • one or more of the active compounds may be admixed or mixed with diluents, binders, dispersants, lubricants, stabilizers, cosolvents, and then formulated into formulations by conventional methods. . These formulations may be applied as a coating agent, or may be applied in two or more coating layers, as needed.
  • the oral liquid compositions include pharmaceutically acceptable liquid solutions, suspensions, emulsions, syrups, medicinal liquors, and the like.
  • one or more of the active compounds can be dissolved, suspended or emulsified in a conventional diluent (e.g., purified water, ethanol or mixtures thereof, etc.).
  • a conventional diluent e.g., purified water, ethanol or mixtures thereof, etc.
  • the above compositions may contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, flavoring agents, preservatives, buffers and the like.
  • Mycophenolate Mofetil or a pharmaceutically acceptable salt thereof has an efficacy against a drug-resistant phenotype influenza virus, thereby providing a martinisol or Use of a pharmaceutically acceptable salt for the preparation of a medicament for combating a phenotype of influenza virus.
  • the anti-drug phenotype influenza virus may be a H1N1 or H3N2 resistant variant virus smear.
  • the drug-resistant phenotype influenza virus is an influenza-resistant influenza virus strain.
  • the invention also provides a method of treating an influenza disease in a subject comprising administering an effective amount of malitol mycophenolate or a pharmacologically acceptable salt thereof to an individual in need thereof.
  • the method of the invention is to administer an effective amount of the drug to an individual infected with H1N1, H3N2 or a resistant strain thereof.
  • the method of the invention is to administer an effective amount of the drug to an individual infected with an influenza virus that is resistant to influenza.
  • treatment refers to the purpose of healing, healing, alleviating, soothing, altering, correcting, ameliorating, ameliorating or affecting the disease, the symptoms of the disease, the disability caused by the disease, or the predisposition to the disease.
  • a composition comprising one or more active agents is administered or administered to an individual having the disease, the symptoms of the disease, or a predisposition to have the disease.
  • the term "effective amount" means that a drug or agent may cause an effect of treating, curing, preventing or ameliorating a disease, disorder or side effect, or reducing a disease or condition, as compared to a corresponding subject who does not receive the amount. Rate of progression.
  • the scope of the term also includes amounts effective to promote normal physiological function.
  • the therapeutically effective amount of a drug or agent depends on a number of factors, such as the age and weight of the animal, the actual condition and severity of the need for treatment, and the dosage form. The nature, and the route of administration are all factors to be considered.
  • matte mycophenolate or a pharmacologically acceptable salt thereof may be used in combination with one or more other anti-influenza agents.
  • the agent may be selected from the group consisting of an M2 inhibitor, an NA inhibitor, a RA polymerase inhibitor, an interferon, and an siRNA.
  • the agent can be (3R,4R,5S)-4-acetamide-5-amino-3-(1-ethylpropoxy)sanecyclohexane-1 - Carboxylic acid (oseltamivir, oseltamivir phosphate; flu, Tamiflu).
  • the combination of the drugs may be used to mix the drugs in the same dosage form, or separately in different dosage forms, such as separate capsules, tablets, troches, injections. It can be administered simultaneously (in parallel) or sequentially.
  • the malic mycophenolate or a pharmacologically acceptable salt thereof is administered orally to the individual simultaneously with oseltamivir.
  • MDCK Madin-Darby canine kidney cells (Madin-Darby dog tubular cell line).
  • FBS Fetal bovine serum
  • TPCK culture medium tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin
  • DMEM Dulbecco' s modified Eagle medium
  • PBS Phosphate-buffered saline
  • Virus Wipe InfluenzaA (HlNl / H3N2) (Source: Prof. Yao Zhenwen, Department of Pathology, Sanjun General Hospital).
  • ELISA reading instrument BIOTEK CERES 900 EIR READER. Evaluation of survival rate of host cells by MTT method
  • Mycophenolate Mofetil can be prepared as follows: Mycophenolic acid 3.2 g (10 mmol) in an ice bath is dissolved in 30 ml of dichloropurine (dichloride) solution. , adding oxalyl chloride 1.5 ml (17.5 mmol) and dimethylformamide (Dimethylformamide) 2 drops, and the mixture was stirred at room temperature for 3 hours. The mixture was evaporated under vacuum to give 11b compound.
  • the preparation reaction process is as follows:
  • the l ib compound was dissolved in 20 ml of ethyl acetate solution, and 4-(2-hydroxyethyl)morpholine (2.6 mg (20 mmol)) was added at room temperature. The mixture was stirred for 30 minutes, and the mixture was evaporated under vacuum to obtain a crude product of maltitol mycophenolate, followed by addition of 50 ml of water and neutralization to pH 7.0 with 0.1 N hydrochloric acid to promote the pure compound of martinisol. Precipitate (4.7 g, 81.0%, mp. 95-96 °C).
  • the antiviral activity assay measures the extent to which a host cell infects a virus by the survival rate of the viral host cell, and the survival rate of the host cell is a method of cell viability assay (MTT assay).
  • MTT assay cell viability assay
  • the principle is that 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, MTT) yellow aqueous solid can be removed from the living cells of the granule gland (mitochondria) Hydrogenase (dehydrogenase) metabolism, the tetrazolium ring is reduced to a purple insoluble precipitate formazan (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-formazan) and accumulated in cells due to living cells
  • the mitochondrial enzyme is catalytically active, so the measured absorbance is proportional to the number of viable cells. Therefore, in this example, the survival rate of the cells was evaluated by using
  • virus strains of H1N1 and H3N2 were selected for the activity test against influenza virus, and the details are as follows:
  • cell culture The cells were thawed and cultured in a 37 ° C, 5 % carbon dioxide incubator. When the cells were over 80%, the cells were washed with phosphate buffer (PBS) and trypsin was added for 5 minutes to attach. In the cultured jni cells, the cells were added to the cell culture medium (DMEM) and reacted with trypsin, centrifuged (1200 rpm, 5 minutes), the supernatant was aspirated, the cells were mixed with a small amount of medium, and the cells were counted and added to the cell culture medium. (DMEM) Dilute to the cell concentration required for the experiment for screening for anti-influenza activity.
  • PBS phosphate buffer
  • trypsin was added for 5 minutes to attach.
  • DMEM cell culture medium
  • DMEM Dilute to the cell concentration required for the experiment for screening for anti-influenza activity.
  • the administration conditions were divided into D + V, D, V, control group (Mock) and blank group (Blank), and cultured at 37 °C, 5 % carbon dioxide incubator (incubator) for 48 hours;
  • D + V Simultaneous administration of 50 ⁇ l of different concentrations of test drug and 50 ⁇ l of influenza virus (0.01 ⁇ );
  • V Give 50 ⁇ l of influenza virus (0.01 ⁇ ) and 50 ⁇ l of TPC medium;
  • Blank Does not contain MDCK cells and TPCK medium.
  • An influenza virus strain of 0.01 MOI (multiplicity of infection) was administered.
  • the drug to be tested has a recovery effect on the apoptosis phenomenon caused by the virus, + + + is an effective drug, and the effective drug must have the same result twice or more.
  • Ribavirin is a control group. Table 2. Minimum inhibitory concentration and drug toxicity of each compound against influenza virus (H3N2)
  • Table 3 shows the inhibitory effects of different concentrations of malitol mycophenolate mofetil (MPM) and its hydrochloride (MPM-N) on WSN influenza virus in the antiviral activity test, and the results showed that the concentration was high (100 g/ml). At low concentrations (0.098 ⁇ g/ml), they have anti-influenza activity.
  • MPM malitol mycophenolate mofetil
  • MPM-N hydrochloride
  • ⁇ Ribavirin is a control group.
  • Example 3 Minimum inhibitory concentration against drug virus
  • influenza strains tested include:
  • HlNl T.R. - H1N1 influenza virus (Tamiflu) resistant virus forest Influenza A virus (H1N1) has been transformed, this strain is similar to Influenza A Taiwan/937/2009 after sequencing.
  • H3N2 - Influenza A strain H3N2 : Approximate to Influenza A/New York/469/2004
  • ⁇ Ribavirin is a control group.
  • EC 5() half effective concentration
  • CC 5 . Half cytotoxic concentration
  • selection index (SI) CC 50 /EC 50 .
  • MPM malt mycophenolate mofetil
  • MPM-N malt mycophenolate mofetil
  • Ribavirin was used to inhibit the Tamiflu-resistant virus strain rWSNH274Y, compared to the malte mycophenolate hydrochloride (MPM-N). Viral infection can be inhibited at a concentration of 5 ⁇ , superior to Ribavirin.
  • MPM- alone or in combination effectively increases the survival rate of influenza virus-infected mice. The survival rate of the mouse model of influenza virus infection is analyzed, and each compound is evaluated. Anti-influenza virus efficacy In this example, the H1N1WSN virus strain was used for animal testing against influenza virus.
  • mice Male 6-week-old BALB/c mice were anesthetized with Zoletil and infected with 1X10 by intranasal injection (in). 3 PFU of the virus, followed by oral administration.
  • the administration conditions were divided into (1) control group: virus infection only; (2) virus infection and administration of Tamiflu, 0.1 mg g for 7 consecutive days; (3) virus infection and administration of Tamiflu, 1 mg/Kg, continuous 7 (4) Viral infection and administration of MPM-N, 0.3 mg for 7 consecutive days; (5) Viral infection and administration of Tamiflu 0.1 mg Kg and MPM-N 0.3 mg for 7 consecutive days.
  • mice The survival rate of the mice was observed after influenza virus infection and the changes in body weight were measured, and the results are shown in Figures 2(A) and (B).
  • the results of the study confirmed that mice who did not take any drug had a survival rate of only 5.4% (2/37) after infection, and if the survival rate of MPM-N mice alone increased to 23.3% (7/30), it was administered alone.
  • the survival rate of mice was 46.7% (14/30). If high doses of Tamiflu (1 mg/Kg) were administered, the survival rate was 100% (13/13).

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Abstract

本发明公开了马替麦考酚酯（Mycophenolate Mofetil）或其药理上可接受的盐类在制备用于抗流感病毒的药物中的用途。本发明还公开了马替麦考酚酯或其药理上可接受的盐类等在制备用于对抗抗药表现型流感病毒的药物中的用途。本发明也涉及一种治疗流感疾病的方法，其包括将有效量的马替麦考酚酯或其药理上可接受的盐类投予有需要的个体。

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马替麦考酚酯或其盐类用于制备抗流感病毒之药物的用途 技术领域
本发明涉及马替麦考酴酯（ Mycophenolate Mofetil )或其药理上可接受的盐类等 在制备用于抗流感病毒的药物中的用途。 背景技术
流行性感冒病毒（简称流感病毒）是人类生命中最具威胁性的病毒之一， 自 20 世纪就发生四次流行性感冒大流行， 分别是 1918~1919年（西班牙流感， H1N1 ) 、 1957年（亚洲流感， H2N2 ) 、 1968年（香港流感， H3N2 )及 1977年（俄罗斯流 感， H1N1 ) 。 其中， 又以 1918年西班牙流感最为严重， 其 H1N1病毒株是由禽流 感以及人类流感病毒突变而成， 在全世界造成 2~4千万人的死亡。 而， 2009年墨西 哥燔发新型 H1N1流感疫情， 已怀疑有一百多万人感染， 造成一万多人的丧命， 目 前也持续蔓延中 ( Neumann, G.; Noda, T.; Kawaoka, Y. Emergence and Pandemic Potential of Swine-Origin H1N1 Influenza Virus. Nature. 459: 931-939, 2009 ) 。 另外， 在 2010年全国流感防控会中提出要特别注意季节性 A型 H3N2流感的侵袭， 此病 毒株也在过去造成不少的伤亡。
流感病毒是一种造成人类与动物患流行性感冒的负链单股 R A病毒， 是属于 正黏液病毒科（Orthomyxoviridae )， 依据病毒核蛋白、 遗传物质、 基质蛋白抗原特 性的不同， 可分为 A、 B、 C三个血清型。 其中， A型流感病毒会导致不同宿主间 的流行性感冒， 因其病毒外表的两种抗原血凝素 （hemagglutinin, HA )及神经胺酸 neuraminidase, NA )为多型性， 前已发现 16种 HA ( Hl~16 )及 9种 NA( N1~N9 ), 可发生遗传性抗原变异； B型流感病毒抗原变异较少只会引起地区性感染， C型流 感病毒主要是以猪为宿主， 对人体的感染较少见， 因此上述所说的世界性大流感就 是由 A型流感病毒所导致的疫情。
A型流感病毒结构自外而内可分为外膜、 基质蛋白及核心三个部分。 外膜有约 500个放射状向外排列的突起， 为上述两种抗原类型： 柱状突起（HA )及蘑菇状突 起（NA ) 。 HA能与多种动物红血球的表面受体吸附引起凝集， 经裂解后可分为重 链及轻链， 使得病毒与宿主细胞相互融合； NA 主要具有水解唾液酸的活性， 切断 病毒与宿主细胞最后的联系， 使病毒从吸附的红血球脱落， 防止病毒的聚集， 促进 在黏液中的移动。 基质蛋白是由 Ml、 M2所组成， 有保护病毒核心及维系病毒的结 构。 另外核心是由 8个负链单股 RNA片段所组成， 其与核蛋白 （NP )及 RNA聚 合酶 （PB1、 PB2及 PA )相结合缠绕成核糖核蛋白体。 而流感病毒之所以会造成 人们的恐慌主要是由于它的变异方式会产生新的亚型而让我们措手不及， 其突变方 式有两种， 一为抗原飘移 （ antigenic drift ) , 其是指流感病毒亚型的抗原 （ΝΑ )胺 基酸序列的点突变所产生小变异； 另一个为抗原转变 （antigenic shift ) , 是流感病 毒变异最常见的类型， 每隔十年就会发生一次抗原性大变异， 造成的原因是由于宿 主同时受到两种不同病毒株感染， 病毒 RNA进行基因重组而产生新的病毒株， 影 响甚大（陈鸿珊， 张兴权， 抗病毒药物及其研究方法， 化学工业出版社， 328-331， 2006. ) 。
流行性感冒大多发生在秋冬、 早春， 其侵袭的目标为呼吸道黏膜上皮细胞， 并 在宿主细胞内繁殖， 导致黏膜充血、 水肿及细胞变性、 脱落等病变。 潜伏期通常 1 至 3天， 开始出现发烧、发冷、 头痛、 鼻塞、 全身酸痛等征状， 当蔓延至下呼吸道， 则可能引起支气管炎和间质性肺炎， 由于流感病毒会降低呼吸道黏膜上皮细胞清除 和黏附异物的能力， 因此经常造成继发性肺炎感染， 是其主要造成流感疾病死亡原 因之一 ( Morens, D. M.; Taubenberger, J. K.; Fauci, A. S. Predominant Role of Bacterial Pneumonia as a Cause of Death in Pandemic Influenza: Implications for Pandemic Influenza Preparedness. J. Infect. Dis. 198: 962― 970, 2008 ) 。
目前， 对于流感的防疫与治疗大多釆取小区隔离及支持性药物治疗， 而使用治 疗预防方式分为疫苗和抗病毒药物。 但是对于日趋变异的病毒， 疫苗防护还是有风 险性； 而目前抗病毒药物主要有三种，第一种为 M2抑制剂（ M2 protein inhibitor )， 作用于病毒穿膜蛋白 M2离子信道， 阻碍 H+进入病毒内部，使得病毒外膜无法与核 内体（endosome ) 融合， 病毒便无法释放 RNA， 这类药物为金刚烷（Adamantan ) 衍生物， 如金刚烷胺 ( amantadine ) 、 金刚乙胺（rimantadine )； 第二种为神经胺酸 酶（NA )抑制剂 （ neuraminidase inhibitors ) ， 作用在神经胺酸酶， 使病毒无法水解 唾液酸，进而无法离开宿主细胞，阻止病毒扩散，这类药物有奥司他韦（oseltamivir, 充流感 )、才 L那米韦（ zanamivir,瑞乐沙）、帕拉米韦（ peramivir )和环成垸（ cyclopentane ) 或11各17定衍生物 ( pyrrolidine derivatives )； 第三种为 RNA聚合醉 ( RNA polymerase ) 抑制剂， 主要抑制 RNA聚合酶（PB1、 PB2、 PA )合成病毒蛋白质的路径， 药物有 2'-脱氧 -2'-氟乌苷（2'-deoxy-2，- fluoroguanosine, FdG ) 、 T-705; 另外也有利用干扰素 及 SiRNA ( small interfering RNAs ) 等治疗方式来防止病毒的感染 （ Clercq, E. D. Antiviral Agents Active against Influenza A Viruses. Nat. Rev. Drug. Discov. 5: 1015-1025, 2006 ) 。
然而， 上述的药物皆无法达到全面抑制各种类型的流感病毒， 且有抗药性及药 物副作用的缺失。 A型流感病毒的抗原变异性大， 传染性强， 每年全球约有 5亿人 感染流感， 常造成很高的死亡率， 对于社会产生巨大的负担和经济损失， 因此积极 开发新的抗流感药物实为一重要课题。 发明内容
本发明首次提出马替麦考酚酯（ Mycophenolate Mofetil )或其盐类等具有对抗流 感病毒的功效。
因此在一方面， 本发明提供马替麦考酚酯或其药理上可接受的盐类等在制备用 于抗流感病毒的药物中的用途。
在本发明的一较佳实施例中， 该药物可接受的盐类等可为盐酸盐、 溴酸盐或有 机酸盐等。
另一方面， 本发明的药物可与一或多种抗流感病毒药剂并用， 其中该药剂选自 由 M2抑制剂、 NA抑制剂、 R A聚合酶抑制剂、 干扰素及 SiRNA所组成的群。
在本发明的一较佳实施例中， 该药剂可为 （3R,4R,5S ) -4-乙酰胺 -5-氨基 -3- ( 1- 乙基丙氧基）-1-环己烷）-1-羧酸（奥司他韦， oseltamivir phosphate;克流感， Tamiflu )。
另一方面， 本发明非可预期的发现马替麦考朌酯或其盐类具有抑制抗药表现型 的流感病毒的功效， 因此， 本发明提供一种马替麦考酴酯或其药理上可接受的盐类 在制备用于对抗抗药表现型流感病毒的药物中的用途。
其中， 该抗药表现型的流感病毒可为 H1N1或 H3N2具抗药性的变种病毒株， 在本发明的一较佳实施例中， 该抗药表现型流感病毒为克流感抗药性流感病毒株。
本发明也提供一种治疗个体流感疾病的方法， 其包括将有效量的马替麦考酚酯 或其药理上可接受的盐类投予有需要的个体。 在一具体实施例中， 本发明的方法是 将有效量的药物投予感染 H1N1、 H3N2或其具抗药性的变种病毒株的个体。在一特 定实施例中， 本发明的方法是将有效量的药物投予感染对克流感具抗药性的流感病 毒株的个体。 根据本发明的方法， 马替麦考酚酯或其药理上可接受的盐类可与其他 一或多种抗流感病毒药剂并用。 本发明的其他特征将经由以下详细说明、 各个具体 实例及申请专利范围而清楚呈现。 附图说明
前文所述以及实施方式可通过附加的图式达到更好的说明效果。 为了加强本发 明的说明， 将适当的实施例的图式列举于此。
图 1为以荧光免疫染色法侦测不同浓度药物对抗抗药性流感病毒株的功效的荧 光免疫染色结果图；
图 2为以小鼠动物模式分析各化合物对抗流感病毒的功效的结果图，其中（ A ) 显示不同条件下小鼠的存活率； （B ) 为小鼠的体重变化图。 具体实施方式
本发明说明中的用词通常具有在本技术领域中、 在本发明内容中、 及各用语所 在的特定内容中的原始意义。
本文所使用的 「一』一词， 如未特别指明， 是指至少一个 （一个或一个以上） 的数量。
本文中所述的 「流感病毒」是为流行性感冒病毒的筒称， 是为一种造成人类与 动物患流行性感冒的负链单股 R A病毒，是属于正黏液病毒科（ Orthomyxoviridae ), 依据病毒核蛋白、 遗传物质、 基质蛋白抗原特性的不同， 可分为 A型流感病毒、 B 型流感病毒及 C型流感病毒，再根据血凝素和神经胺酸酶的抗原性分为不同的亚型。 根据世界卫生组织（ WHO )流感病毒株的命名包含 6个要素： 型另 ' 宿主 /分离地区 / 毒株序号 /分离年份（HnNn )， 其中对于人类流感病毒， 省略宿主信息， 对于乙型和 丙型流感病毒省略亚型信息。 本发明中所称的流感病毒包括 A型流感病毒、 B型流 感病毒及 C型流感病毒。 在一特定实施例中， 流感病毒特别为 H1N1、 H3N2或其 具抗药性的变种病毒株， 尤指对克流感 （Tamiflu )具抗药性的变种病毒株。
根据本发明， 未可预期地发现马替麦考酚酯（ Mycophenolate Mofetil )或其药物 可接受的盐类等具有对抗流感病毒的功效， 因此提供马替麦考酚酯（Mycophenolate Mofetil )或其药理上可接受的盐类在制备用于抗流感病毒的药物中的用途。
本文中所述的 「马替麦考盼酯 （Mycophenolate Mofetil )」 是指 2-吗啉乙基 (E)-6-(l,3-二氢 -4-羟基 -6-甲氧基 -7-曱基 -3-氧代 -5-异苯并呋喃) -4-甲基 -4-己烯酸乙酯 (2-morpholinoethyl(E)-6-(l,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzo iuranyl)-4-methyl-4-hexenoate)， 具有下列结构式：
Figure imgf000007_0001
本文所使用的 「药理上可接受的盐类」是指对于人类或哺乳动物服用具安全性 且有效的盐类化合物， 具有其所需的生物活性。 药物上可接受的盐类包括但不限于 本发明的马替麦考盼酯 （ Mycophenolate Mofetil ) 的酸性或碱性盐类， 例如与盐酸、 氢溴酸、 碘酸、 硝酸、 硫酸、 硫酸氢钠、 磷酸、 磷酸酯、 醋酸、 乳酸、 水杨酸、 柠檬酸、 酒石酸、 泛酸、 重酒石酸、 抗坏血酸、 丁二酸、 马来酸、 富马酸、 葡萄糖 酸、 曱酸、 苯甲酸、 谷氨酸、 甲基磺酸、 对曱苯磺酸合成的碱性盐； 或与铝、 钙、 锂、 镁、 钾、 钠、 锌和二乙醇胺盐合成的碱性盐。
在本发明的一较佳具体实施例中， 该药理上可接受的盐可为盐酸盐。
另一方面， 该药物可与一或多种抗流感病毒的药剂并用， 包含但不限于 M2抑 制剂 （ M2 protein inhibitor ), 如金刚烷衍生物的金刚烷胺（ amantadine )与金刚乙胺 ( rimantadine ); 神经胺酸酶（ NA )抑制剂， 如有奥司他韦（ oseltamivir, 克流感）、 札那米韦 ( zanamivir, 瑞乐沙 )、 帕拉米韦 ( peramivir )和环戊坑 ( cyclopentane ) 或17各 4汙生物 （ pyrrolidine derivatives ); RNA聚合酶 ( R A polymerase )抑制剂， 如 2'-脱氧 -2'-氟鸟苷（ 2'-deoxy-2'-fluoroguanosine, FdG )、 T-705;以及干扰素或 SiRNA 等。
在本发明的另一较佳具体实施例中，前述抗流感病毒的药剂为克流感（ Tamiflu )， 即（3R,4R,5S)-4- 乙酰胺 -5-氨基 -3-(1- 乙基丙 氧基）-1-环 己垸）-1-羧酸 ((3 R,4 , 5 S)-4-acetamido-5 -amino-3 -(1 -ethy lpropoxy )- 1 -cy clohexane- 1 - carboxylic acid; oseltamivir phosphate).根据本发明，该药物可以包括但不限于非经肠道或口服投药。 非经肠道投药的药物其形式包括溶液、 悬浮液、 乳液， 及可在使用前刻溶解或悬浮 于溶剂中的固体可注射组合物。 可通过溶解、 悬浮或乳化一或多种活性成分于稀释 剂中来制备该注射液。前述稀释剂的实例为用于注射的蒸馏水、生理盐水、植物油、 醇类及其组合。 又， 该注射液可含有安定剂、 助溶剂、 悬浮剂、 乳化剂、 平滑剂、 缓冲液、 保存剂等。 该等注射液是在最终制剂步骤中灭菌或以无菌程序制备。 本发 明的药物亦可被制剂成无菌固体配制品， 例如， 通过冷冻干燥， 并可在使用前刻灭 菌或溶解于无菌可注射水或其他无菌稀释剂。 该药物亦可经口投药， 其中该组合物 13 000336
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可为固体或液体形式。 固体组合物包括锭剂、 丸剂、 胶嚢、 分散性粉剂、 颗粒及其 类似物。 口服组合物亦包括漱口药及舌下片剂。 胶嚢包括硬胶嚢及软胶嚢。 在此类 口服固体组合物中， 一或多种活性化合物可自行混合， 或与稀释剂、 结合剂、 散解 剂、 润滑剂、 安定剂、 助溶剂混合， 接着以习知方法制剂成配制品。 当需要时， 此 等配制品可以涂布剂涂布， 或可以二或多种涂布层涂布。 另一方面， 口服液体组合 物包括医药上可接受的液态溶液、 悬浮液、 乳液、 糖浆、 药酒， 及类似物。 在此类 组合物中，一或多种活性化合物可被溶解、悬浮或乳化在通用稀释剂中（如纯化水、 乙醇或其等的混合物等等）。 除了此类稀释剂， 前述组合物亦可含有润湿剂、 悬浮 剂、 乳化剂、 甜味剂、 调味剂、 香料、 保存剂及緩沖液及其类似物。
根据本发明， 未可预期地发现马替麦考酚酯（ Mycophenolate Mofetil )或其药物 可接受的盐类具有对抗抗药表现型流感病毒的功效， 因此提供一种马替麦考朌酯或 其药理上可接受的盐类在制备用于对抗抗药表现型流感病毒的药物中的用途。
其中， 该抗药表现型的流感病毒可为 H1N1或 H3N2具抗药性的变种病毒抹， 在本发明的一较佳实施例中， 该抗药表现型流感病毒为克流感抗药性流感病毒株。
本发明也提供一种治疗个体流感疾病的方法， 其包括将有效量的马替麦考酚酯 或其药理上可接受的盐类投予有需要的个体。 在一具体实施例中， 本发明的方法是 将有效量的药物投予感染 H1N1、 H3N2或其具抗药性的变种病毒株的个体。在一特 定实施例中， 本发明的方法是将有效量的药物投予感染对克流感具抗药性的流感病 毒抹的个体。
本文所使用的 「治疗』一词是指为了治愈、 愈合、 减轻、 舒緩、 改变、 矫正、 改善、 改进或影响该疾病、 该疾病的症状、 该疾病引起的残疾或罹患该疾病的倾向 的目的， 而将包含一或多种活性剂的组合物施用或投与至患有该疾病、 该疾病的症 状或有罹患该疾病的倾向的个体。
本文所使用的 「有效量』一词是指相较于未接受该量的对应对象， 药物或药剂 可造成治疗、 治愈、 预防或减轻疾病、 疾患或副作用的效果， 或是降低疾病或疾患 的进展速率。 该词的范围亦包含对促进正常生理功能有效的量。 药物或药剂的治疗 上有效量取决于多个因素。 例如动物的年龄及体重、 需要治疗的实际情况及严重程 度、 剂型的本质、 及投药途径等皆为须考虑的因素。
本文所使用 「对象」、 「个体」 和「病患」 三个词汇可交互使用， 是指任何需要 进行治疗或医疗的哺乳类动物， 特别是指人类。 其他的对象可能包括牛、 狗、 猫、 天竺鼠兔、 大鼠、 小鼠和马等。 根据本发明的方法， 马替麦考酚酯或其药理上可接受的盐类可与其他一或多种 抗流感病毒药剂并用。 该药剂可选自由 M2抑制剂、 NA抑制剂、 R A聚合酶抑制 剂、 干扰素及 SiRNA 所组成的群。 在本发明的一较佳实施例中， 该药剂可为 ( 3R,4R，5S ) -4-乙酰胺 -5-氨基 -3- ( 1-乙基丙氧基）小环己烷） -1-羧酸（奥司他韦， oseltamivir phosphate; 克流感， Tamiflu )。 根据本发明， 药物的并用可以将药物混合 于相同剂型， 或分别置于不同剂型中， 如分开的胶嚢、 片剂、 锭剂、 注射剂。 可同 时 （并行）或依序施用。 在一具体实施例中， 马替麦考酚酯或其药理上可接受的盐 类是与奥司他韦同时以口服投予个体。
无须进一步的阐述， 咸相信本发明所属技术领域中具有通常知识者基于前述说 明即可利用本发明至最广的程度。 下述实施例仅仅是作为例示说明的用， 而非以任 何方式限制其余的揭露内容。
【实施例 1
细胞药理实验及免疫染色材料
细胞林 ( Cell line ): MDCK: Madin-Darby canine kidney cells ( Madin-Darby 氏狗肾小管细胞株）。
细胞培养液：
1. 10% Fetal bovine serum ( FBS );
2. TPCK培养液 ( tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin );
3. DMEM ( Dulbecco' s modified Eagle medium )。
细胞生理緩冲液： Phosphate-buffered saline ( PBS )。
病毒抹： InfluenzaA ( HlNl / H3N2 ) (来源：姚振文教授，三军总医院病理部）。 ELISA读数测定仪： BIOTEK CERES 900 EIR READER。 以 MTT方法评估宿 主细胞的存活率
动物实验材料
雄性 6周龄的 BALB/c小鼠是购自乐斯科生物科技股份有限公司 实施例 1. 马替麦考酚酯（ Mycophenolate Mofetil, MPM )及其盐酸盐（ MPM-N ) 的制备
马替麦考盼酯（ Mycophenolate Mofetil )可依下述方法制备： 于冰浴中的麦考朌 酸 ( mycophenolic acid ) 3.2 g ( 10 mmol )溶于 30 ml的二氯曱垸 ( dichloromethane ) 溶液中， 加入草酰氯 （oxalyl chloride ) 1.5 ml ( 17.5 mmol ) 及二甲基甲酰胺 ( dimethylformamide ) 2滴， 在室温下搅拌该混和液 3小时。 真空下蒸发该混和液 而得 11 b化合物。 制备反应流程如下：
Figure imgf000010_0001
将 l ib化合物溶于 20 ml的乙酸乙酯 （ ethyl acetate )溶液中， 加入 4- ( 2-羟乙 基 )吗啉（ 4- ( 2-hydroxyethyl ) morpholine ) 2.6 ml ( 20 mmol )， 在室温下搅拌该 混和液 30 分钟， 真空下蒸发该混和液而得马替麦考酚酯粗产物， 接着添加 50 ml 水并以 0.1 N 盐酸中和至 pH 7.0以促进马替麦考朌酯纯化合物的沉淀（ 4.7 g, 81.0%, mp. 95-96 °C )。
马替麦考酚酯的盐酸盐（ Mycophenolate Mofetil hydrochloride )可依下述方法制 备： 于室温下将马替麦考酚酯（ Mycophenolate Mofetil ) 2 g ( 4.61 mmol )溶于 50 ml 的乙酸乙酯溶液中， 搅拌该溶液并加入 0.3 ml ( 1.2当量） 的醋酸及 0.7 ml ( 1.2当 量） 的三甲基氯硅烷（ trimethylchlorosilane ), 搅拌该混和液 1 小时并过滤沉淀物， 将固态物质于室温下以乙酸乙酯洗涤三次并于真空下蒸发而获得 2.11 g的马替麦考 酚酯盐酸盐 （97.6%，mp. 157.2 °C )。 实施例 2. 纯化物的最小抑制浓度及药物毒性实验
抗病毒活性试验是以病毒宿主细胞的存活率来评估宿主细胞感染病毒的程度, 而宿主细胞的存活率是以细胞存活率分析 （MTT assay ) 的方法。 原理为四甲基偶 氮峻盐 （3- ( 4,5-dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide, MTT )黄色 水溶液固体可被活细胞的粒腺体 ( mitochondria ) 中的脱氢酵素 （ dehydrogenase ) 代谢， 将 tetrazolium ring 还原 呈紫 色 不溶性沉淀物 formazan ( 3- ( 4,5-dimethylthiazol-2-yl ) -2,5-diphenyl-formazan )而堆积在细胞中， 因活细胞的粒 线体酵素才具有催化活性， 故所测得的吸光值会与活细胞数量成正比关系， 因此本 实施例是利用 formazan产量的多寡来评估细胞的存活率。
具体地说明， 本实施例选用 H1N1及 H3N2的病毒株进行抗流感病毒的活性试 验， 其细节如下：
一、 细胞培养 细胞解冻后培养在 37 °C、 5 %二氧化碳培养箱中， 等至细胞长满八成左右时， 以磷酸緩冲液 ( PBS ) 清洗细胞， 加入胰蛋白腾（trypsin )反应 5 分钟， 使贴附在 培养 jni的细胞脱落，加入细胞培养基（ DMEM )中和 trypsin的反应，离心（ 1200 rpm、 5分钟），吸除上清液，以少量培养基混勾细胞，计数细胞后加入细胞培养液（ DMEM ) 稀释成实验所需的细胞浓度， 以便进行抗流感病毒活性筛选。
二、 实验步骤
1. 将细胞稀释至指定浓度 ( 2 X 104 细胞 /孔）植入％孔板（ 96-well plate )中， 于 37 °C、 5 %二氧化碳培养箱 ( incubator )培养 20-24小时；
2.每个孔内的细胞以 100 u l 的细胞生理緩冲液（PBS ) 清洗两次， 最后一次 加入 100 μ g孔的 TPCK培养液， 置入培养箱， 待测药物稀释完成后再给药；
3. 给药条件分为 D + V、 D、 V、 对照组（Mock )和空白组（Blank )给药， 于 37 °C、 5 %二氧化碳培养箱 ( incubator )培养 48小时；
4. 两天后以显微镜观察细胞凋亡情形；接着进行 MTT检测，分别在 D + V、D、 V、 Mock和 Blank组中加入 MTT试剂 20 μ 1 ( 5 mg/ml ), 等待 5小时；
5. 吸去培养液， 在 D + V、 D、 V、 Mock和 Blank中加入 25 μ 1 glycine緩冲液 以及 100 μ 1二甲基亚讽 ( dimethyl sulfoxide, DMSO )。
6. 进行读盘， 测量波长 540 nm处的吸光值。
三、 给药条件
1. D + V: 同时给予 50 μ 1不同浓度待测药物和 50 μ 1流感病毒（ 0.01 ΜΟΙ );
2. D: 给予 50 μ 1不同浓度待测药物和 50 μ 1 TPC 培养液；
3. V： 给予 50 μ 1流感病毒（ 0.01 ΜΟΙ )和 50 μ 1 TPC 培养液；
4. Mock: 含 MDCK细胞和 100 μ 1 TPCK培养液；
5. Blank: 不含 MDCK细胞和 TPCK培养液。
四、 流感病毒（H1N1 / H3N2/WSN )感染量
施予 0.01 MOI ( multiplicity of infection ) 的流感病毒株。
五、 待测药物浓度
纯化物：100 g/ml、50 μ g/mK 25 μ g/ml、 12.5 μ g/mL 6.25 μ g/ml、 1.563 μ g/ml、 0.391 g/ml及 0.098 μ g/ml八种浓度。
六、 阳性对照组
抗病毒药物 Ribavirin
七、 MTT检测结果读取 1. 细胞存活率:
( D + V / D / V ) - Blank
X
Mock
2.0 ~ 25 % 细胞存活率 （ cell survival ) 纪录为 + / -；
25〜 50 % cell survival纪录为 +；
50 ~ 75 % cell survival纪录为 + +；
75〜 100 % cell survival纪录为 + + +；
> 100 % cell survival纪录为 + + + +。
其中， 待测药物对于病毒造成细胞凋亡现象有回复作用者， + + +以上为有效 药物， 且有效药物必须有两次或以上的相同结果。
请参阅表 1， 在抗 H1N1流感病毒活性试验中， 马替麦考酚酯 （MPM )及其盐 酸盐（MPM-N ) 由高浓度 ( 100 g/ml )至低浓度 ( 0.391 μ g/ml ) 同时给予待测药 物与 H1N1流感病毒时， 均可使 MDCK细胞存活率达 75%， 显示具有抗流感病毒 活性， 并且对细胞无药物毒性。 表 1、 各化合物抗流感病毒（H1N1 ) 的最小抑制浓度及药物毒性
MPM MPM-N Ribavirin: k
D+V D D+V D D+V D
100 μ g/ml +++ +++ +++ ++++ ++++ ++++
50 μ g/ml +++ ++屮 +++ +++ ++++ ++++
25 μ g/ml +++ +十+ +++ +++ ++++ ++++
12.5 μ g/ml +++ +++ +++ +++ ++++ ++++
6.25 μ g/ml +++ +++ +++ +++ +++ ++++
1.563 μ g/ml +++ +++ +++ +++ + ++++
0.391 μ g/ml ++++ ++++ 十 +++ ++++ + ++++
0.098 μ g/ml ++ ++++ ++ ++++ + ++++
* Ribavirin 为对照组。 表 2、 各化合物抗流感病毒（H3N2 )的最小抑制浓度及药物毒性
MPM MPM-N Ribavirin
D+V D D+V D D+V D
100 μ g/ml +/- ++++ +/- ++++ ++++ ++++
50 U g/ml +++ +++ +++ ++++ ++++ ++++
25 μ g/ml +++ +++ +++ ++++ ++++ ++++
12.5 μ g/ml +++ +++ +++ ++++ ++++ ++++
6.25 μ g/ml +++ +++ ++++ ++++ +++ ++++
1.563 μ g/ml +++ +++ +++ +++ 十 /- ++++
0.391 μ g/ml + ++++ + ++++ +/- ++++
0.098 μ g/ml +/- ++++ 十 /- ++++ +/- ++++
*Ribavirin是为对照组。 此外， 表 3为抗病毒活性试验中不同浓度的马替麦考酚酯（MPM )及其盐酸盐 ( MPM-N )对于 WSN流感病毒的抑制效果， 结果显示由高浓度 ( 100 g/ml )至 低浓度 ( 0.098 μ g/ml ) 均具有抗流感病毒活性。
MPM MPM-N Ribavirin*
D+V D D+V D D+V D
100 μ g/ml ++++ ++++ ++++ ++++ ++++ ++++
50 μ g/ml ++++ ++++ ++++ ++++ ++++ ++++
25 μ g/ml ++++ ++++ ++++ ++++ ++++ ++++
12.5 μ g/ml ++++ ++++ ++++ ++++ +++ ++++
6.25 μ g/ml ++++ ++++ ++++ ++++ ++++ ++++
1.563 μ g/ml ++++ ++++ ++++ ++++ ++++ ++++
0.391 μ g/ml ++++ ++++ ++++ ++++ + +++
0.098 μ g/ml ++++ +++ ++++ +++ + +++
^Ribavirin是为对照组。 实施例 3. 对抗药性病毒的最小抑制浓度
依前述方法检测下列化合物针对流感病毒的抗药变种的最小抑制浓度 ( MIC ): MPM: 马替麦考酚酯
MPM-N: 马替麦考酚酯的盐酸盐
受测流感病毒株包括：
HlNl T.R. - H1N1克流感 （Tamiflu )抗性病毒林： A型流感病毒（H1N1 )经 变种， 此一病毒株经定序后近似于 Influenza A Taiwan/937/2009
H3N2 - A 型流感病毒株 （ H3N2 ) : 经定序后近似于 Influenza A/New York/469/2004
WSN - Influenza A/WSN/33 ( HlNl ): 经定序后近似于 Influenza A/Hon kong/470/97
Influenza B - B型流感病毒抹
HlNl - A型流感病毒株 ( HlNl ) 表 4、 各化合物的半数有效浓度、 半数细胞毒性浓度及选择指数的比较
HlNl T.R. HlNl WSN H3N2
化合物 条件 S.I.
EC50 EC50 EC50
Ribavirin* D+V 3.125 3.125 12.5 >16
MPM D+V 0.391 0.098 1.560
Dl+V 0.391 0.098 1.560
Vl+D 0.391 0.098 1.560
D >200 >200 >200 >2000
MPM-N D+V 0.391 0.049 1.560
Dl+V 0.391 0.049 1.560
Vl+D 0.391 0.049 1.560
D >200 〉200 >200 >4000
^Ribavirin 为对照组。 EC5(): 半数有效浓度， CC5。： 半数细胞毒性浓度， 选择指数 ( S.I. ): CC50/EC50。 表 4的结果显示马替麦考酚酯 （MPM )及其盐酸盐 （MPM-N )对于具有抗药 性的各流感病毒株其最小抑制浓度均优于对照组的 Ribavirin 表 5、 各化合物对具抗药性的流感病毒株最小抑制浓度的比较
Figure imgf000015_0001
根据表 5的结果证实， 马替麦考酚酯 （MPM)及其盐酸盐 （MPM-N)具有对 抗具抗药性的流感病毒株的功效。 实施例 4. MPM-N有效抑制 Tamiflu抗药性流感病毒株 rWSNH274Y
为评估马替麦考酚酯盐酸盐（ MPM-N )对于抗药性流感病毒抹的功效， 使用细 胞荧光免疫染色法侦测病毒核蛋白以确认是否有效抑制病毒感染。 具体地说明， 本 实施例选用对 Tamiflu具有抗药性的 H1N1病毒株 rWSNH274Y进行试验。
请参阅图 1, 如荧光免疫染色的结果所示， 需使用 100 μ M的 Ribavirin以抑制 Tamiflu抗药性病毒株 rWSNH274Y, 相较于此， 马替麦考酚酯盐酸盐 （MPM-N) 的浓度达 5 μΜ即可抑制病毒感染， 优于 Ribavirin 实施例 5. MPM- 的单独或合并使用有效增加流感病毒感染小鼠的存活率 以流感病毒感染的小鼠动物模式分析存活率， 评估各化合物抗流感病毒的功效 本实施例选用 H1N1WSN 病毒株进行抗流感病毒的动物试验， 将雄性 6 周大的 BALB/c小鼠以舒泰 ( Zoletil )麻醉后以鼻腔内注射（i.n.) 的方式感染 1X103 PFU 的病毒， 而后以口服给药。 给药条件分为 （ 1 )控制组： 仅病毒感染； （2) 病毒感 染并投予 Tamiflu, 0.1 mg g, 连续 7天； (3 )病毒感染并投予 Tamiflu, 1 mg/Kg, 连续 7天； （4 )病毒感染并投予 MPM-N, 0.3 mg, 连续 7天； （ 5 )病毒感染并投予 Tamiflu 0.1 mgKg及 MPM-N 0.3 mg, 连续 7天。
于流感病毒感染后观察小鼠的存活率并测量体重变化，其结果如图 2( A )及（ B ) 所示。 研究结果证实， 未服用任何药物的小鼠于感染后存活率仅余 5.4% (2/37), 若单独投予 MPM-N小鼠存活率提高至 23.3% (7/30), 单独投予低剂量的 Tamiflu (0.1 mg/Kg), 小鼠存活率为 46.7% ( 14/30)， 若投予高剂量的 Tamiflu ( 1 mg/Kg) 其存活率可达 100% ( 13/13 ), 而当合并投予低剂量 Tamiflu ( 0.1 mg/Kg )及 MPM-N ( 0.3 mg )时， 小鼠存活率大幅提升为 93.3% ( 28/30 )。 亦即通过单独投予或合并他 种药物使用， MPM-N均展现对抗流感病毒的绝佳功效。
虽然本发明以较佳实施例揭露如上， 然其并非用以限定本发明， 任何熟习本发 明技术者， 当可在不脱离本发明的精神和范围内， 作些许的更动与润饰， 则应属本 发明申请专利范围所界定的保护范围。

Claims

权利要求
1. 马替麦考酚酯（ Mycophenolate Mofetil )或其药理上可接受的盐类在制备用 于抗流感病毒的药物中的用途。
2. 如权利要求 1所述的用途， 其中该药理上可接受的盐类为盐酸盐。
3. 如权利要求 1所述的用途， 其中该流感病毒为 H1N1、 H3N2或其具抗药性 的变种病毒株。
4. 如权利要求 1 所述的用途， 其中马替麦考盼酯或其药理上可接受的盐类可 与一或多种抗流感病毒药剂并用， 该药剂选自由 M2抑制剂、 NA抑制剂、 RNA聚 合酶抑制剂、 干扰素及 SiR A所组成的群。
5. 如权利要求 4所述的用途， 其中该药剂为 （ 3R，4R，5S ) -4-乙酰胺 -5-氨基 -3- ( 1-乙基丙氧基）小环己烷）小羧酸（磷酸奥司他韦）。
6. 马替麦考酴酯（ Mycophenolate Mofetil )或其药理上可接受的盐类在制备对 抗抗药表现型流感病毒的药物的用途。
7. 如权利要求 6所述的用途， 其中该药理上可接受的盐类为盐酸盐。
8. 如权利要求 6所述的用途， 其中该抗药表现型流感病毒为 H1N1或 H3N2 具抗药性的变种病毒株。
9. 如权利要求 6所述的用途，其中该抗药表现型流感病毒为对（ 3R，4R,5S ) -4- 乙酰胺 -5-氨基- 3- ( 1-乙基丙氧基） -1-环己烷）小羧酸 (磷酸奥司他韦）具抗药性 的流感病毒株。
10. 一种治疗个体流感疾病的方法， 其包括将有效量的马替麦考酚酯或其药理 上可接受的盐类投予有需要的个体。
11. 如权利要求 10所述的方法， 其中该个体感染 H1N1、 H3N2或其具抗药性 的变种病毒株。
12. 如权利要求 10所述的方法， 其中该个体感染对 （3R,4R，5S ) -4-乙酰胺 -5- 氨基 -3- ( 1-乙基丙氧基） -1-环己烷） -1-羧酸（磷酸奥司他韦）具抗药性的流感病毒 株。
13. 如权利要求 10所述的方法，其中马替麦考酚酯或其药理上可接受的盐类是 与一或多种抗流感病毒药剂并用， 该药剂选自由 M2抑制剂、 NA抑制剂、 R A聚 合酶抑制剂、 干扰素及 SiRNA所组成的群。
14. 如权利要求 10所述的方法，其中马替麦考酚酯或其药理上可接受的盐类是 与 （3R,4R,5S ) -4-乙醜胺 -5-氨基 -3- ( 1-乙基丙氧基） -1-环己烷） -1-羧酸（磷酸奥 司他韦） 并用。
PCT/CN2013/000336 2013-03-22 2013-03-22 马替麦考酚酯或其盐类用于制备抗流感病毒之药物的用途 WO2014146218A1 (zh)

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