WO2013131496A1 - 治疗病毒疾病的成分和方法 - Google Patents

治疗病毒疾病的成分和方法 Download PDF

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WO2013131496A1
WO2013131496A1 PCT/CN2013/072402 CN2013072402W WO2013131496A1 WO 2013131496 A1 WO2013131496 A1 WO 2013131496A1 CN 2013072402 W CN2013072402 W CN 2013072402W WO 2013131496 A1 WO2013131496 A1 WO 2013131496A1
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virus
cells
infection
enterovirus
pcr
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PCT/CN2013/072402
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English (en)
French (fr)
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艾德铭
任培君
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中国科学院上海巴斯德研究所
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Priority to US14/384,075 priority Critical patent/US9872875B2/en
Priority to EP13757343.2A priority patent/EP2875829A4/en
Priority to IN8266DEN2014 priority patent/IN2014DN08266A/en
Priority to JP2014560239A priority patent/JP6116596B2/ja
Priority to SG11201405559TA priority patent/SG11201405559TA/en
Priority to KR1020147027450A priority patent/KR20150005526A/ko
Publication of WO2013131496A1 publication Critical patent/WO2013131496A1/zh

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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
    • A61K31/70—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
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic 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
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • 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
    • 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

Definitions

  • the present invention is in the field of biomedicine; more specifically, the present invention relates to ingredients and methods for treating viral diseases. Background technique
  • Hand, foot and mouth disease is a common viral infection in the western Pacific and is a major cause of illness and death among children in China and Asia.
  • two million children in the Western Pacific suffered from hand, foot and mouth disease (more than 1.6 million in China, more than 340,000 in Japan, and more than 110,000 in Vietnam).
  • temperate climate countries have occurred in the summer and early fall every two to three years (Solomon, Lewthwaite et al. 2010). Since 2008, China's mainland has exploded every spring and summer.
  • Hand, foot and mouth disease mainly affects children under five years of age.
  • the main symptoms include oral ulcers, hand, foot and mouth herpes, and burning of the mouth (herpes angina).
  • Most cases have only mild symptoms and are self-limiting.
  • there are still a large number of cases (more than 27,000 in China in 2010, about 1.6%) showing severe neurological symptoms such as aseptic meningitis, encephalitis and polio-like paralysis and central nervous system disorders, and Neurogenic pulmonary edema and cardiac dysfunction (Huang, Liu et al. 1999; Yang, Wang et al. 2009; Weng, Chen et al. 2010; Rhoades, Tabor-Godwin et al. 201 1).
  • 2010 and 201 there were 905 and 506 deaths in China each.
  • Hand, foot and mouth disease is caused by non-polio enterovirus.
  • the main pathogenic viruses were EV71 and CVA16 (Yan, Gao et al.; Zhang, Wang et al. 2010; Zhu, Zhu et al. 2010) , most of the severe cases and deaths are caused by EV71 infection.
  • the EV71 C4 subtype is the main epidemic strain, while in other parts of Asia there are EV71 C l, C2, C4 and B3, and the B4 subtype is prevalent (Yang, Ren et al. 2009).
  • the CVA16 virus-infected drug is controlled from the source. Summary of the invention
  • a P2X receptor antagonist for the preparation of a suppressing sense single strand A composition of an RNA virus picornavirus.
  • the P2X receptor antagonist is for use in the preparation of a composition for preventing, ameliorating or treating hand, foot and mouth disease.
  • the composition is further for: systemic or parenteral administration; preferably, the composition is for oral, intravenous, intramuscular or inhalation administration.
  • a method of inhibiting a positive single-stranded RNA virus picornavirus comprising: administering an effective amount of a P2X receptor antagonist to a subject in need of inhibition of the virus.
  • the subject is a virus-infected mammal, including a human enterovirus 71 or a Coxsackie-infected person, a monkey or a mouse, and more preferably a hand, foot and mouth disease patient.
  • the P2X receptor antagonist is selected from the group consisting of PPADS, iso-PPADS, PPNDS,
  • the virus is an enterovirus.
  • the virus is enterovirus A.
  • the virus is human enterovirus type 71 and coxsackie virus.
  • the virus is human enterovirus type 71, C4 subtype and coxsackievirus A16 subtype.
  • FIG. 1 RD cells were infected with EV71 clinical isolates at different E02 concentrations with a M0I of 0.1. After incubation for 46 hours, the viral RNA in the culture supernatant was extracted, and the relative virus amount of EV71 was determined by quantitative RT-PCR. The numbers in columns 2, 3, and 4 represent the results of 3 replicates, and the graph on the right is the average of three results.
  • RD cells were infected with EV71 diluted with 10 times at different E02 concentrations. After 3-4 days of culture, CPE (cytopathic phenomenon) was observed under light microscope, and stained with crystal violet to calculate half of tissue culture infection dose (TCID 50 ). .
  • FIG. 3 RD cells cultured in 12-well plates were infected with EV71 at different E02 concentrations. After 5 to 7 days of culture, plaque forming units (PFU) were calculated by staining.
  • PFU plaque forming units
  • FIG. 4 RD cells were infected with EV71 MAV diluted 10 times at different E02 concentrations. After 3 to 4 days of culture, CPE was observed under a light microscope, and stained with crystal violet to calculate half of the tissue culture infection dose (TCID 5() ).
  • FIG 6. RD cells were infected with Coxsackievirus A16 diluted 10 times at different E02 concentrations. After 2 days of culture, CPE was observed under a light microscope, and stained with crystal violet to calculate half of the tissue culture infection dose (TCID 5() ).
  • Figure 7. Three-day-old newborn ICR mice were infected with EV71 clinical isolates. Blood samples were collected 5 days after infection; viral RNA was extracted from serum, and viral load was detected by real-time quantitative RT-PCR.
  • FIG. 1 Distribution of CVA16 in various organs or tissues of ICR newborn mice.
  • No. 1 and No. 2 indicate blood samples taken from two separate mice.
  • FIG. 9 Three-day-old neonatal ICR mice were infected with CVA16 virus. Tissues were taken 6 days after infection, blood was collected for blood collection, tissue and serum RNA were extracted, CVA16 virus RNA was determined, and viral load was calculated.
  • Cytoter Glo reagent was used to determine the cytotoxicity of different concentrations of E02 for RD cells.
  • FIG. 11 Three-day-old ICR mice were intraperitoneally injected with 50 ⁇ l of 50 mg/kg, 20 mg/kg E02 or 50 ⁇ l of DMEM medium for 4 consecutive days, daily injection, to observe the toxicity results.
  • Figure 14 mRNA expression of six P2X isoforms in RD cells by RT-PCR.
  • Figure 15. E02 inhibits replication of EV71 isolates SH-TS and SH-RS in RD cells.
  • FIG. 20 P2X receptor antagonists inhibit EV71 replication in RD cells.
  • the inventors have intensively studied for the first time to disclose a new use of a P2X receptor antagonist for preparing a virus composition.
  • the P2X receptor antagonist exerts a prophylactic or therapeutic effect on hand, foot and mouth disease by inhibiting the virus.
  • P2X is a family of ATP-gated ion channel proteins expressed in cell membranes, typically homologous or heterotrimeric, consisting of one extracellular domain, two transmembrane regions, and two intracellular regions.
  • the P2X family consists of seven subtypes. Triggered by extracellular ATP, the P2X ion channel is opened, causing calcium influx, intracellular calcium ion aggregation, and downstream signal transduction by MAPK, PKC and calmodulin. CErb, Liao et al. 2006).
  • viruses which are viruses of the sense single-stranded RNA virus picornavirus family, including Aphtho virus, Avian Avihepato virus, Cardio virus, Taylor disease Theilo virus, Enterovirus, Erbo virus, Hepato virus, Kobuvirus, Parechovirus, etc.; more preferably, the virus is Enterovirus genus, including enterovirus AH, rhinovirus AC; more preferably, the virus is enterovirus A, including Baboon enterovirus , human Coxsackie virus A2-8, A10, A12, A14, A16, human enterovirus 71, 76, 89, 90-92, etc.; more preferably, enterovirus 71 (EV71) (including A, B and C subtypes), Coxsackie virus (including class A and B, preferably including A16 subtype; CVA16).
  • viruses which are viruses of the sense single-stranded RNA virus picornavirus family, including Aphtho virus, Avian Avihepato virus, Cardio virus, Taylor disease Theil
  • enterovirus 71 or coxsackievirus A16 subtype infection is a major cause of hand, foot and mouth disease.
  • the Ministry of Health's Guidelines for the Diagnosis and Treatment of Hand, Foot and Mouth Disease (2010 Edition) and a number of published studies have indicated that EV71 and CVA16 are the main pathogens. Therefore, P2X receptor antagonists can be used to prevent hand, foot and mouth disease.
  • the P2X receptor is a P2X1-7 subtype receptor.
  • the P2X receptor antagonist is selected from the group consisting of PPADS, Iso-PPADS, PPNDS, Suramin, NF023 (Suramin derivative TNP-ATP, NF279, NF 157, Evans Blue.
  • the P2X receptor antagonist may be: Suramin, PPADS, iso-PPADS, PPNDS, NF023, NF279, TNP-ATP, NF157, Evans Blue.
  • the present invention also includes isomers, racemates, pharmaceutically acceptable salts, hydrates, precursors, derivatives or the like of the various compounds enumerated above, as long as the isomers, exogenous
  • the rot, the pharmaceutically acceptable salts, hydrates, derivatives or analogs also have the function of inhibiting viruses such as enterovirus 71 or coxsackievirus A16 subtype.
  • the “isomers” include: conformational isomers, optical isomers (e.g., enantiomers and diastereomers), geometric isomers (e.g., cis and trans isomers).
  • derivative or analog refers to a compound having the structural formula similar to the above-exemplified P2X receptor antagonists, particularly having the same parent core structure, but some of the compound groups are replaced by similar groups.
  • the compound still retains the function of suppressing viruses such as enterovirus 71 or coxsackievirus A16 subtype.
  • H is substituted by C1-C4 alkyl; C1 alkyl is substituted by C2 alkyl; substitution occurs between halogen groups (including F, Cl, Br, I); OH, OCH 3 , OCH 2 CH 3 , OCH 2 Substitution occurs between CH 3 and the like.
  • the "pharmaceutically acceptable salt” means a salt formed by reacting the above compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal.
  • These salts include, but are not limited to: (1) salts with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with the following organic acids, such as acetic acid , lactic acid, citric acid, succinic acid, fumaric acid, gluconic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, tartaric acid, maleic acid, or refined ammonia acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid,
  • salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, ammonium or water-soluble amine salts such as N-methylglucamine salts, and lower alkanolammonium salts.
  • other pharmaceutically acceptable amine salts such as methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine
  • a salt an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trishydroxyethylamine salt, and an amine salt formed from morpholine, piperazine, and lysine, respectively, or other conventional "prodrugs"" form.
  • Compound The object has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures
  • the "precursor of a compound” means that the precursor of the compound is converted into a P2X receptor antagonist compound, or a P2X receptor, enumerated in the foregoing by metabolic or chemical reaction in a patient after administration by an appropriate method.
  • Precursors of the compounds include, but are not limited to, carboxylates, carbonates, phosphates, nitrates, sulfates, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds, phosphoramides of the compounds. , glucoside, ether, acetal, etc. Sieve method
  • the inventors' research has found that a class of P2X receptor antagonists can inhibit viral replication, which may be exerted by targeting P2X receptors.
  • a P2X receptor antagonist can exert a drug that binds to a P2X receptor based on this characteristic by selectively binding to a P2X receptor to exert a virus-inhibiting action. Thereafter, a drug that is truly useful for suppressing the virus can be found from the substance.
  • the present invention provides a method of screening for a potential drug for controlling hand, foot and mouth disease, the method comprising: (1) providing a system comprising (eg, expressing) a P2X receptor; (2) administering a candidate substance to the system of step (1) To observe the binding of the candidate substance to the P2X receptor; if the candidate substance can bind to the P2X receptor, the candidate substance is a potential drug for preventing hand, foot and mouth disease.
  • the system comprising (e.g., expressing) a P2X receptor may be, for example, a cell system, and the cell may be a cell which endogenously expresses a P2X receptor; or may be a cell which recombinantly expresses a P2X receptor.
  • the system comprising the P2X receptor may also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal such as a mouse, a rabbit, a sheep, a monkey, etc. ) Wait. Detection of whether a compound binds to a receptor or a partial subunit of a receptor is a technique known in the art.
  • a control group in the screening, in order to make it easier to observe the difference in the binding of the P2X receptor to the candidate substance, a control group may be provided, and the control group may be included without adding the candidate substance.
  • System of P2X receptors in order to make it easier to observe the difference in the binding of the P2X receptor to the candidate substance, a control group may be provided, and the control group may be included without adding the candidate substance.
  • the candidate substance may include, but is not limited to, a small molecule compound, a polypeptide, a ligand.
  • the candidate substance is a small molecule compound.
  • the candidate substance can be a variety of P2X receptor antagonists.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for inhibiting the virus.
  • the invention also includes a virus-inhibiting substance obtained by the screening method.
  • the compound of the present invention can be used to prepare a pharmaceutical composition.
  • the pharmaceutically acceptable carrier can be a solid or a liquid.
  • the solid preparation includes a powder, a tablet, a pill, a capsule, a sachet, a suppository and a dispersible granule.
  • the solid carrier can be one or more. They can be used as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.
  • the carrier is a finely divided solid, and the compound of the invention is present in the mixture with the finely divided active component.
  • the compound is mixed with the desired binding carrier in an appropriate ratio and compressed to the desired shape and size.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, carboxymethylcellulose, sodium carboxymethylcellulose, low melting waxes and cocoa butter.
  • cachets or lozenges, tablets, powders, capsules, pills, cachets, and lozenges may be solid dosage forms suitable for oral administration.
  • Formulations in solution form include solutions, suspensions and emulsions, such as water or aqueous propylene glycol solutions.
  • solutions for parenteral injection liquid preparations, it can be prepared in an aqueous polyethylene glycol solution.
  • the aqueous solution suitable for oral administration can be prepared by dissolving the active ingredient in water and adding a suitable coloring agent, flavoring agent, emulsifier and thickening agent as needed.
  • composition can be used for systemic administration, topical administration or parenteral administration, and the like.
  • Aqueous suspensions suitable for oral administration can be prepared by dissolving the finely divided active component in aqueous viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium hydroxymethylcellulose, and other well known suspensions. Agent.
  • the compound for use in the present invention is administered at a starting dose of from 0.01 mg to 200 mg/kg body weight per day, more preferably from 0.25 mg to 100 mg/kg body weight, more preferably from 0.5 mg to 50 mg/kg body weight.
  • these dosages may vary depending on the needs of the patient, the severity of the condition being treated, and the compound employed. In general, treatment begins with a smaller dose that is less than the optimal dosage of the compound, after which a small increase in dose is achieved. Good results, for convenience, the total daily dose can be subdivided into divided doses within one day if needed.
  • compositions of the invention may also be used in combination with other therapeutic agents or adjuvants.
  • the P2X receptor antagonist or the composition containing the P2X receptor antagonist of the present invention may be placed in a kit for sale or use.
  • the P2X receptor antagonist or the composition containing the P2X receptor antagonist can be placed in a kit form and placed in a kit.
  • "Unit dosage form” means a dosage form required for preparing a P2X receptor antagonist or a composition containing a P2X receptor antagonist of the present invention in a single administration for convenience of administration, including but not limited to various solid agents (such as tablets). Agent), liquid agent, capsule, sustained release agent.
  • Agent such as tablets
  • the composition of the unit dosage form of 1-3 may be administered daily.
  • the kit further includes instructions for administering a P2X receptor antagonist to a subject in need of suppression of a virus (such as a patient suffering from a viral disease, or a place containing a virus) to know that the person is correctly administering the drug.
  • a virus such as a patient suffering from a viral disease, or a place containing a virus
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the manufacturer's recommended conditions. . Percentages and parts are calculated by volume unless otherwise stated. I. Experimental materials 1, cell line
  • Rhabdomyosarcoma cells were purchased from ATCC, number VR-805.
  • the cell culture medium was DMEM containing 10% (v/v) FBS, and a 1% (w/v) ampicillin/streptomycin solution was added. 2, the virus strain
  • EV71 clinical isolate EV71 FY 573, isolated from the sample of hand-foot-mouth cases in Anhui province, China in 2008, with genotype EV71 C4 and Genbank accession number HM_064456. Provided by the Shanghai Institute of Life Sciences, Chinese Academy of Sciences.
  • the EV71 clinical isolates SH-TS and SH-RS were provided by the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, and the genotype was C4.
  • the EV71 clinical isolate SEP-4 isolated and sampled from the Cambodian clinical hand, foot and mouth disease sample by the Institut Pasteur of Cambodia, has a genotype of C4.
  • EV71-FY23 strain Provided by the Virus Immunology Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences, batch number: 20121001, Genebank accession number EU812515.1.
  • EV71 M.A.V. EV71 mouse adapted strain
  • EV71 FY 573 was repeatedly passaged in newborn mice.
  • CVA16 Coxsackie virus A16 subtype, shzh05-l (GenBank# EU262658).
  • E02 was purchased from Sigma-Aldrich and administered as Suramin Sodium.
  • the suramin used in the monkey experiment was supplied by Bayer and administered as Suramin free acid.
  • iso-PPADS (0683-10 mg), NF 279 (1199-lOmg), PPNDS (1309) were purchased from Tocris.
  • EV71 bow positive: CCCTGAATGCGGCTAATC (SEQ ID NO: 1);
  • EV71 probe FAM 6-carboxyfluorescein
  • AACCGACTACTTTGGGTGTCC GTGTTTC SEQ ID NO: 3
  • TAMRA 6-carboxytetramethylrhodamine
  • GAPDH forward bow I substance GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 4);
  • GAPDH reverse primer GAAGATGGTGATGGGATTTC (SEQ ID NO: 5);
  • P2X1-164F CCTCTTCGAGTATGACACC (SEQ ID NO: 6);
  • P2X1-164R CAGAGACACTGCTGATGAG (SEQ ID NO: 7);
  • P2X2-202F CTGGACATGCTGGGAAACG (SEQ ID NO: 8);
  • P2X2-202R TGCCCTTGGAGAAGTGGAAT (SEQ ID NO: 9);
  • P2X3-153F GGCTCGACAGCGTTTCT (SEQ ID NO: 10); P2X3-153R: TGCCAGCATTCCCGTAT (SEQ ID NO: 11);
  • P2X4-201F TGGGATGTGGCGGATTAT (SEQ ID NO: 12);
  • P2X4-201R TACGCACCTGCCTGTTGAGA (SEQ ID NO: 13);
  • P2X5-220F TCTTTGCCTGGTGCCCGTTG (SEQ ID NO: 14);
  • P2X5-220R ATCACGGAGCCCAGTCGGAAG (SEQ ID NO: 15);
  • P2X6-205F GGAGGACAAAGTATGAGGAGG (SEQ ID NO: 16);
  • P2X6-205R GAATGGGTTGGCAAGTGG (SEQ ID NO: 17);
  • P2X7-175F CGTGGAGAAGTGAAGAAG (SEQ ID NO: 18);
  • P2X7-175R TCGGTCAGAGGAACAGAGC (SEQ ID NO: 19).
  • DMEM Invitrogen cat# 11965118;
  • FBS Invitrogen cat# 10099141;
  • Virus RNA extraction kit QIAamp Viral RNA Mini Kit, QIAGEN cat#52906;
  • Total RNA extraction kit for cells RNeasy Mini Kit (250), QIAGEN cat#74106;
  • Tissue preservation solution RNAstore sample preservation solution, Tiangen Biotechnology (Beijing) Co., Ltd., DP408-02
  • Tissue RNA extraction kit RNAprep pure animal tissue total RNA extraction kit, Tiangen Biotechnology (Beijing) Co., Ltd., DP43 ;
  • Taqman real time RT-PCR reagent ABI taqMan® One-Step RT-PCR, ABI cat#
  • SYBR real time RT-PCR reagent QIAGEN QuantiTect SYBR Green RT-PCR Kit (200), Cat# 204243;
  • RT-PCR reagent QIAGEN OneStep RT-PCR Kit (100); Cat# 201212;
  • DNA electrophoresis agarose Biowest Argarose, Cat# BW-R0100;
  • DNA marker DL2,000 DNA Marker, Cat# D501;
  • Crystal violet Santa Cruz cat# sc-207460;
  • CPE cytopathic effect, cytopathic effect
  • CT cycle threshold, cycle threshold
  • EC50 50% effective concentration, half effective agent EV71 : Enterovirus 71 , Enterovirus 71;
  • HFMD Hand Foot and Mouth Disease, Hand, Foot and Mouth Disease
  • iso-PPADS pyridoxal-phosphate-6-azophenyl-2' , 5 '-disul honic acid;
  • M.A.V. Mouse Adapted Virus, mouse adapted to the virus
  • MAPK Mitogen-activated protein kinases, mitogen-activated protein kinase
  • MOI Multiplicity of infection, multiplicity of infection
  • NF 023 8,8'-[carbonyl3 ⁇ 4z'5(imino-3,l-phenylenecarbonylimino)]3 ⁇ 4z5-l,3,5-naphthalene-trisulphonic acid, hexasodium salt;
  • PKC Protease kinase C, protein kinase C;
  • PPADS pyridoxal-phosphate-6-azophenyl-2', 4'-disul honic acid; polyphosphate- 6-benzene azo- 2', 4'-disulfonic acid;
  • PPNDS Pyridoxal-5 '-phosphate-6-(2 ' -naphthylazo-6 '-nitro-4 ' ,8 '-disulfonate) tetrasodium salt, ⁇ -5 '-phosphate-6-(2'-naphthylazo-6' -nitro-4', sodium 8'-sulfonate) tetrasodium salt;
  • RD cell Rhabdomyosarcoma cell, rhabdomyosarcoma cells;
  • T.O.A. Time of Addition assay, adding time experiment
  • TCID 50 50% Tissue Culture Infective dose, half tissue culture infection dose
  • TNP-ATP trinitrophenol-ATP, picric acid-ATP
  • UTP uridine triphosphate, uridine triphosphate
  • EV71 and CVA16 are the main pathogens causing hand, foot and mouth disease, and the inventors have determined in the following examples the ability of E02 (Suramin Sodium) or its analogs to inhibit EV71 and CVA16 infection in vitro and infection in vivo.
  • E02 Suramin Sodium
  • E02 inhibits virus EV71 clinical isolate (EV71 FY 573) is infected with RD cells (in vitro test) 1.
  • E02 can inhibit the replication of EV71 in RD cells.
  • RD cells were infected with EV71 clinical isolate (EV71 FY 573) at different E02 concentrations with a MOI (multiplicity of infection) of 0.1.
  • the virus RNA in the culture supernatant was extracted after 46 hours of culture, and the relative virus amount of EV71 was determined by quantitative RT-PCR. The detailed steps are as follows:
  • Drug pre-incubation virus Dilute the virus stock solution to 5 ⁇ 10 4 TCID 50 /ml in DMEM, dispense 100 ⁇ l per well into 96-well plate, and add 11 ⁇ l of the corresponding concentration of E02 or DMEM dissolved in DMEM. base,
  • b. Drug pre-incubation of cells Discard the cell culture medium in 96-well plate, add 100 ⁇ l of DMEM per well, add 11 ⁇ l of the corresponding concentration of E02 solution or DMEM medium, and incubate at 37 ° C for 1 hour;
  • Virus harvesting and RNA extraction After incubating the infected cells for 46 hours, transfer 140 ⁇ l of the supernatant to a 96-well deep well plate and extract the viral RNA using the viral RNA extraction kit (QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906). , according to the standard operating procedures of the kit;
  • Viral load assay The EV71 5' UTR gene was detected in an ABI 7900HT 384-well plate PCR system using the ABI Taqman one-step RT-PCR kit (ABI TaqMan® One-Step RT-PCR, cat# 4309169). The reaction system and PCR procedures are shown in Tables 1 and 2. EV71 5 'UTR quantitative RT-PCR thermal cycle program (Taqman);
  • CT value of the PCR was converted to the viral load using a PCR standard curve (the standard curve was diluted by a ten-fold gradient of the virus of known titer, RNA was extracted, and CT value was determined by RT-PCR).
  • Table 1 EV71 5 'UTR real-time quantitative RT-PCR reaction system (Taqman)
  • RD cells were infected with EV71 (clinical isolate EV71 FY 573) diluted with 10 times at different E02 concentrations. After 3-4 days of culture, CPE (cytopathic phenomenon) was observed under light microscope, and stained with crystal violet to calculate half of tissue culture infection. dose. The detailed steps are as follows:
  • Drug pre-incubation virus Dilute the virus stock solution in 10-fold gradient with DMEM medium, dispense 100 ⁇ l per well into 96-well plate, add 11 ⁇ l of E02 dissolved in DMEM, incubate at 37 °C. Hour
  • b. Drug pre-incubation of cells Discard the cell culture medium in 96-well plate, add 100 ⁇ l of DMEM to each well, add 1 1 ⁇ l of the corresponding concentration of E02 solution, and incubate at 37 ° C for 1 hour;
  • E02 could inhibit CPE caused by EV71 virus infection in RD cells, and E02 reduced the half effective dose of EV71 TCIDso in RD cells to 4.7 micromolar, as shown in Fig. 2. Therefore, E02 significantly reduced EV71 TCID 50 in RD cells.
  • E02 reduces EV71 plaque forming unit in RD cells
  • RD cells cultured in 12-well plates were infected with EV71 (clinical isolate EV71 FY 573) at different E02 concentrations, and plaque forming units were counted after 5 to 7 days of culture. The detailed steps are as follows:
  • Drug pre-incubation virus Dilute the virus stock solution to 50000 PFU/ml in DMEM medium 10 fold.
  • b. Drug pre-incubation of cells Discard the cell culture medium in a 12-well plate, add 500 ⁇ l of DMEM to each well, add 56 ⁇ l of the corresponding concentration of E02 solution, and incubate at 37 ° C for 1 hour;
  • E02 was able to inhibit the number of plaques formed by EV71 in RD cells.
  • E02 decreased the half effective dose of EV71 PFU in RD cells to 2.6 ⁇ M, see Figure 3. Therefore, E02 can reduce EV71 plaque forming units in RD cells.
  • E02 can inhibit EV71 clinical isolates infected with RD cells: inhibit EC50 replication of EV71 in RD cells is 6.93 micromolar; reduce RD cells In TCID 5 .
  • the EC50 is 4.7 micromolar; the EC50 of the reduced PFU is 2.6 micromolar.
  • Example 2 E02 inhibits enterovirus EV71 murine adaptation strain infected with RD cells (in vitro test) 1, E02 reduces EV71 MAV TCID 50 in RD cells
  • RD cells were infected with 10-fold gradient dilution of EV71 MAV at different E02 concentrations. After 3 to 4 days of culture, CPE was observed under a light microscope, and stained with crystal violet to calculate half of the tissue culture infection dose. The specific steps are the same as the corresponding TCID 5Q test steps in Embodiment 1.
  • RD cells cultured in 12-well plates were infected with EV71 M.A.V. at different E02 concentrations, stained for 5 to 7 days, and stained to form plaque forming units.
  • the detailed procedure is the same as the corresponding PFU test method in Example 1.
  • E02 decreased the half effective dose of EV71 M.A.V. PFU in RD cells to 2.0 micromolar, see Figure 5. therefore,
  • E02 reduces EV71 M.A.V. PFU in RD cells.
  • TCID50 and PFU tests showed that E02 inhibited mice from adapting to EV71 infection with RD cells with EC50 of 8.07 micromolar and 2.0 micromolar, respectively.
  • Example 3 E02 inhibits coxsackievirus A16 (CVA16) infection of RD cells (in vitro test)
  • RD cells were infected with Coxsackievirus A16 diluted at a 10-fold gradient at different E02 concentrations. After 2 days of culture, CPE was observed under a light microscope, and stained with crystal violet to calculate half of the tissue culture infection dose. The specific steps are the same as those of the TCID 5 Q test method in Example 1.
  • E02 reduced CVA16 TCID 50 in RD cells.
  • the results of the above Examples 1-3 indicate that E02 can inhibit EV71 and CVA16 infection of RD cells in vitro.
  • the EC50 of E02 inhibited EV71 clinical isolates was less than 6.93 micromolar, and 10 micromolar E02 completely inhibited the infection of RD cells by E02 clinical strain and mouse adapted strain, and decreased the TCID 5Q of CVA16 in RD cells.
  • Example 4 E02 inhibits enterovirus EV71 infection in ICR newborn mice
  • mice Three-day-old newborn ICR mice were infected with EV71 clinical isolate (EV71 FY 573), and the ability of E02 to inhibit EV71 in mice was tested.
  • the experimental mice were divided into three groups: drug group, infection group, and placebo group.
  • the drug group was intraperitoneally injected with 50 ⁇ l of 50 mg/kg E02, and the infected group and the placebo group were intraperitoneally injected with 50 ⁇ l of DMEM medium.
  • the drug group was intraperitoneally injected with 50 mg/kg doses of E02 and 5 X.
  • the EV71 and GAPDH RNA load ratios were calculated, with an average of 15.0 in the infected group and 1.8 in the drug group, as shown in Figure 7.
  • the results showed that E02 reduced the amount of EV71 virus in the serum of mice, and 50 mg/kg of E02 could reduce the amount of virus in the serum of ICR newborn mice infected with EV71.
  • Example 5 E02 inhibits Coxsackie virus A16 infection ICR newborn mice
  • TCIDso CVA16 virus 50 ⁇ l was injected into two newborn mice of three-day-old ICR, and serum and tissues were collected 5 days after infection (brain, bone marrow, heart, small intestine, kidney, liver, lung, muscle, spleen). , thymus).
  • Extraction of serum viral RNA (viral RNA extraction kit, QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906) o Tissue placed in RNAstore sample preservation solution (; Tiangen, DP408-02) stored in -80 ° C refrigerator, with tissue Tissue RNA was extracted from the RNA extraction kit (RNAprep pure animal tissue total RNA extraction kit).
  • the CVA16 virus genome and GAPDH gene copy were detected in a ABI 7900HT 384-well PCR system using a one-step real-time quantitative RT-PCR kit (QIAGEN QuantiTect SYBR Green RT-PCR Kit). Primer with EV71 5 'UTR Primers, reaction systems and PCR procedures are shown in Tables 3 and 4. The relative viral amount in each tissue and serum is shown in Figure 8. It can be seen that the CVA16 virus is mainly detected in the brain, spinal cord, muscle and serum.
  • mice Three-day-old newborn ICR mice were infected with CVA16 virus, and the ability of E02 to inhibit EV71 in mice was tested.
  • the experimental mice were divided into three groups: the drug group, the infection group, and the placebo group.
  • the drug group was intraperitoneally injected with 50 ⁇ l of 50 mg/kg E02
  • the infected group and the placebo group were intraperitoneally injected with 50 ⁇ l of DMEM medium.
  • the drug group was intraperitoneally injected with 50 mg/kg doses of E02 and 1 X.
  • the cytotoxicity of different concentrations of E02 to RD cells was determined by Celltitr Glo reagent as follows:
  • E02 cultured cells discard the medium in the cell culture plate, add 180 ⁇ l of DMEM medium containing 2% FBS, and add 20 ⁇ l of the corresponding concentration of E02 DMEM solution, set at 37 ° C, 5% C0 2 Incubator culture; (3) After 46 hours of culture, the ATP level of the cells was measured with PROMEGA Celltitr Glo reagent and determined according to the reagent instructions.
  • E02 is not toxic to ICR newborn mice.
  • E02 inhibits enterovirus EV71 clinical isolate SH-TS and SH-RS infection RD cells 1. E02 inhibition EV71 SH-TS and SH-RS replicate in RD cells
  • RD cells were infected with EV71 SH-TS or SH-RS strain at different E02 concentrations, MOI 0.1. Training 46 After the hour, the viral RNA in the culture supernatant was extracted, and the relative virus amount of EV71 was determined by quantitative RT-PCR. For the specific steps of quantitative RT-PCR, see Example 1.
  • E02 could inhibit the replication of EV71 clinical isolate SH-TS and SH-RS in RD cells.
  • SH-TS 7.14 micromolar could reduce EV71 replication by 10 fold, and 25.35 micromolar could reduce EV71 replication by 100-fold.
  • SH-RS 4.41 micro-molding can reduce EV71 replication by a factor of 10; 16.29 micro-molar can reduce EV71 replication by a factor of 1000, as shown in Figure 15.
  • E02 inhibits replication of EV71 SH-TS and SH-RS in RD cells.
  • RD cells cultured in 12-well plates were infected with EV71 SH-TS or SH-RS strains at different E02 concentrations, and plaque forming units were counted after 5 to 7 days of culture.
  • the detailed procedure is the same as in Example 1, "3, E02 reduces the EV71 plaque forming unit in RD cells".
  • E02 decreased the half effective dose of EV71 SH-TS PFU in RD cells to 2.24 micromolar, and 3.47 micromolar for SH-RS, as shown in Fig. 16.
  • E02 inhibited RD71 infection in EV71 clinical isolates SH-TS and SH-RS.
  • Example 8 E02 inhibits enterovirus EV71 Cambodia isolate SEP-4 infection Vero cells
  • Vero cells, MOI 0.1, were infected with EV71 SEP-4 strain (provided by Institut Pasteur of Cambodia, Cambodia strain) at different E02 concentrations.
  • the virus RNA in the culture supernatant was extracted after 46 hours of culture, and the relative virus amount of EV71 was determined by quantitative RT-PCR. See Example 1 for specific steps.
  • E02 could inhibit the replication of EV71 SEP-4 strain in Vero cells, 5.89 micromolar E02 could reduce EV71 replication by 10 fold, and 15.49 micromolar reduced EV71 replication by 100-fold, as shown in Figure 17.
  • Suramin 50mg/kg was injected intravenously and infected with 4.5 lgCCID 50 EV71 FY-23 virus (EV71-FY23 strain (provided by the Institute of Medical Immunology, Chinese Academy of Medical Sciences) 1 day after the first injection of suramin, Lot number: 20121001, Content: 7.5 lgCCID50/ml, Storage conditions: -80°C
  • the virus load in the adult rhesus monkey was detected daily, and the clinical symptoms and body temperature of the infected animals were observed.
  • Therapeutic and preventive effect of drugs on EV71 infection The specific experimental procedure is as follows:
  • Intravenous challenge was performed according to 4.5 lgCCID50 EV71 FY-23.
  • the body temperature is measured daily, and the animal's appearance and signs, behavioral activities, mental state, abnormality of the administration site, etc. are observed, and the viral load is detected. It was administered intravenously (50 mg/kg surami or saline) at 1, 3, and 5 days after infection.
  • the body temperature was measured at the late stage of infection (21 and 28 days), and the animal's appearance, physical activity, mental state, abnormality of the administration, and the like were observed to detect the viral load.
  • Extract whole blood RNA Take 0.2ml, add 0.8ml TRNzol-A + , mix well and let lOmin at room temperature; add 0.2ml chloroform, cover the tube cover, violently shake for 15sec, let stand at room temperature for 5min, centrifuge at 12500rpm, 4°C 20min, take the water phase 0.4ml in another new tube, add an equal volume of isopropanol, mix, let stand for 30 minutes at room temperature; centrifuge at 12500 rpm for 20 min at 4 ° C, discard the supernatant, add 1 ml of 75% ethanol to wash the precipitate, 12500 rpm, Centrifuge at 4 °C for 30 min. Discard the supernatant, leave it at room temperature for 30 min, dry the RNA, and add 20 ⁇ l of RNase-free ddH20 to fully dissolve the RNA.
  • the viral load was measured and the method of determination was as follows:
  • Standard dilution Standard extraction (according to TaKaRa MiniBEST Viral RNA/DNA extraction Kit Ver. 3.0 instructions), take ⁇ extracted standard, 10 times serial dilution to 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10°
  • Downstream bow I (10 ⁇ ) 5 '-atccagtcgatggctgctca-3 ';
  • the body temperature of the control group showed a certain increase 1 day after infection, and then decreased to about the normal range (about 3 days after infection); the body temperature of the animals in the administration group was basically within the normal range, as shown in Fig. 19.
  • RD cells in 96-well plates were infected with EV71 clinical isolate EV71 FY573 of MOI 10, and EV71 replication was determined by adding 32 ⁇ M E02 (dissolved in DMEM) or DMEM medium before infection, in the three stages of infection and infection. To determine the stage of action of E02 to inhibit EV71 infection. The detailed steps are as follows:
  • Virus harvesting and RNA extraction After incubating the infected cells for 18 hours, transfer 140 ⁇ l of the supernatant to a 96-well deep well plate using a viral RNA extraction kit (QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906) Viral RNA is extracted and performed according to the standard operating procedures of the kit.
  • a viral RNA extraction kit QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906
  • Viral load assay The EV71 5' UTR gene was detected in the ABI 7900HT 384-well plate PCR system using the ABI Taqman one-step RT-PCR kit (ABI taqMan® One-Step RT-PCR, Cat# 4309169). The reaction system and PCR procedures are shown in Tables 1 and 2.
  • the PCR value of the PCR was converted to the viral load using a PCR standard curve (the standard curve was diluted by a virus gradient of known titer, RNA was extracted, and CT value was determined by RT-PCR).
  • E02 is an antagonist of the receptor P2X and is capable of inhibiting P2X1, 2, 3 and 5 subtypes (Khakh, Burnstock et al. 2001; Burnstock 2004; Coddou, Yan et al. 2011) receptors, ie P2X1, 2, Antagonists of the 3 and 5 subtype receptors.
  • P2X is a family of ATP-gated ion channel proteins expressed in the cell membrane, usually homologous or heterotrimeric, consisting of one extracellular domain, two transmembrane regions, and two intracellular regions. The P2X family includes seven subtypes.
  • P2X ion channel Triggered by extracellular ATP, the P2X ion channel is opened, causing calcium influx, intracellular calcium ion aggregation, and downstream signal transduction through MAPK, PKC and calmodulin (Erb, Liao et al. 2006).
  • P2X receptors are widely distributed in higher animal tissues. CValera, Hussy et al. 1994), expressed in various parts of the CNS, triggered by synapses in neurons, transmitted by sensory signals (such as pain, taste, hearing, etc.), smooth muscle contraction, heart Both vascular system blood pressure control and inflammatory response play a role (Surprenant and North 2009).
  • EV71 infection of the central nervous system can cause acute flaccid paralysis, acute transmission myelitis and acute transverse myelitis, can also cause aseptic meningitis and encephalitis, enterovirus infection may also cause potential behavior and memory disorders (Yang, Wang et al. 2009; Rhoades, Tabor-Godwin et al. 2011).
  • EV71 causes neurological disorders mainly by inducing CNS inflammation in patients with viral infection.
  • EV71 infection causes inflammation in the cerebral cortex, brain stem and spinal cord. Patients with EV71 infection often die of pulmonary edema or hemorrhage, and studies have shown that EV71 pulmonary edema is neurogenic (Solomon, Lewthwaite et al. 2010).
  • the inventors used a chemical probe (a known P2X inhibitor or activator) to determine the role of the P2X receptor in EV71 infection.
  • a chemical probe a known P2X inhibitor or activator
  • PPADS is an analog of E02 (Khakh, Burnstock et al. 2001; Burnstock 2004; Coddou, Yan et al. 2011), and the inhibition of P2X subtypes is consistent with E02.
  • RD cells were infected with EV71 clinical isolate FY 573 at different concentrations of PPADS, and the inhibitory effect of PPADS on EV71 replication was determined. The detailed procedure is the same as "1" in the first embodiment. The cytotoxicity of each concentration of PPADS on RD cells was determined, and the detailed procedure was the same as "1" in Example 6.
  • RNA extraction from cells Take 2 X 10 6 RD cells, and extract total RNA using QIAGEN RNeasy kit. The experiment was carried out according to the kit instructions, and the product was dissolved in 50 ⁇ l of nuclease-free water;
  • PCR - step method RT-PCR amplification of each P2X subtype and GAPDH mRNA, reaction system and cycle procedures are shown in Tables 5 and 6.
  • Electrophoresis 2% gel electrophoresis was used to detect the size of the PCR product.
  • Electrophoresis results showed that except for P2X7, the other six P2X subtypes were expressed in RD cells.
  • P2X receptor antagonist inhibits EV71 infection RD cells
  • the inhibition of EV71 replication by 0.1, 1, 10 and 100 micromolar P2X receptor antagonists and agonists was determined.
  • the specific procedure is as follows: "1" in Example 1, but the cells are incubated for 3 to 4 days after infection, optical microscopy. Observe and stain with crystal violet to determine the presence or absence of CPE (cytopathic phenomenon). The cytotoxicity of each concentration of the compound on RD cells was measured. After RD cells were cultured in different concentrations of compounds for 3 to 4 days, they were observed by light microscopy and stained with crystal violet to observe the destruction of the cell layer. A compound that protects cells from infection, has no CPE and does not destroy healthy cell layers, and inhibits EV71 replication in RD cells.
  • P2X receptor 1 2 3 4 5 6 2/3 inhibits EV71 complex
  • Agonist -: inactive, + : activation concentration >1 ⁇ , ++: activation concentration 1 ⁇ ⁇ , +++: activation concentration ⁇ l ⁇ M;
  • the effects of other antagonists and agonists on EV71 are shown in Table 9 and Figure 21.
  • Antagonist 1 2 3 4 5 6 7 P2Y lOg!o Lower IC90 ltimer
  • V V V activation concentration ⁇ 10 nM
  • V V activation concentration 10 nM-300 nM live concentration >300 nM, inactive.
  • V V V activation concentration ⁇ 1 ⁇ M For Agonists: V V V activation concentration ⁇ 1 ⁇ M, V V activation concentration 1-10 ⁇ ⁇ , V activation concentration >10 ⁇ ⁇ . Therefore, ⁇ 2 ⁇ 1 ⁇ 6 subtypes have mRNA expression in RD cells, and multiple P2X receptor antagonists can inhibit RD71 infection in RD cells, and bind P2X signal transduction and P2X abnormalities in relation to disease, suggesting P2X infection in EV71. It plays an important role, and the role of P2X and EV71 is closely related to the pathology of hand, foot and mouth disease caused by EV71 infection. In summary, the following conclusions can be drawn:
  • the P2X receptor antagonist compound E02 is capable of inhibiting EV71 virus infection of RD cells; (2) Compound E02 can reduce the infectivity of Coxsackievirus A16-infected RD cells;
  • Compound E02 can reduce the amount of EV71 virus in serum in ICR newborn mice;
  • Compound E02 can reduce the amount of CVA16 virus in brain, bone marrow, muscle and serum in ICR newborn mice;
  • E02 inhibits the replication of EV71 in rhesus monkeys and inhibits the increase in body temperature of rhesus monkeys caused by EV71.
  • RD cells (6) mRNA of six subtypes of P2X receptors 1 to 6 can be detected in RD cells;
  • Rhoades RE, JM Tabor-Godwin, et al. (201 1). "Enterovirus infections of the central nervous system.” Virology 411(2): 288-305. Solomon, T., P. Lewthwaite, et al. (2010). "Virology, epidemiology, pathogenesis, and control of enterovirus 71.” The Lancet Infectious Diseases 10(11): 778-790.

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Abstract

本发明涉及治疗病毒疾病的成分和方法。首次揭示了一种P2X受体拮抗剂的新用途，用于制备一种治疗病毒感染疾病的组合物。所述的P2X受体拮抗剂通过抑制病毒发挥预防或治疗手足口病的作用。

Description

治疗病毒疾病的成分和方法
技术领域
本发明属于生物医药领域； 更具体地， 本发明涉及治疗病毒疾病的成分和方法。 背景技术
手足口病是西太平洋地区常见的一种病毒性感染疾病， 是引起中国和亚洲地区儿童 疾病及死亡的一大原因。 201 1 年， 西太平洋地区有两百万儿童患手足口病 (中国大于一 百六十万， 日本大于三十四万， 越南大于十一万)。 在过去二十年中， 温带气候国家每两 至三年就有一次发生于夏季和初秋的疫情爆发 (Solomon, Lewthwaite et al. 2010)。 而中 国大陆地区自 2008年后， 每年春夏季都有爆发。
手足口病主要感染五岁以下儿童， 主要症状包括口腔溃疡， 手足和口部疱疹， 及口 部疼痛灼烧 (疱疹性咽峡炎）。 大多数病例只有轻度症状并有自限性。 然而， 依然有大量 病例 (2010年中国两万七千多例，约 1.6%)显示出严重神经系统症状，例如无菌性脑膜炎， 脑炎和脊髓灰质炎样的麻痹及中枢神经系统紊乱以及神经源性肺水肿及心脏功能失常 (Huang, Liu et al. 1999; Yang, Wang et al. 2009; Weng, Chen et al. 2010; Rhoades, Tabor-Godwin et al. 201 1)。 2010年和 201 1年在中国各有 905和 506例死亡病例。
手足口病由非脊髓灰质炎肠道病毒引起， 在 2008年安徽省爆发的手足口病疫情中， 主要致病病毒为 EV71和 CVA16(Yan， Gao et al.； Zhang, Wang et al. 2010; Zhu, Zhu et al. 2010) , 其中绝大部分重症和死亡病例均由 EV71感染引起。 在中国 EV71 C4亚型是 主要流行株， 而在亚洲其他地区有 EV71 C l， C2， C4及 B3， B4亚型的流行 (Yang, Ren et al. 2009)。
手足口病人与人间传播由直接接触感染者口鼻分泌物， 疱液和粪便引起 (Solomon, Lewthwaite et al. 2010)。 感染者在发病后一周的传染性最强， 但症状好转后仍有持续感 染性 (Han， Ma et al. 2010)。 很多感染者 (包括多数成年感染者)并无症状， 出现口腔疱疹 的儿童通过临床和后续病毒学诊断较易诊断。
目前尚无手足口病疫苗及药物。 卫生， 支持治疗， 缓解疼痛， 漱口水和静脉注射免 疫球蛋白是治疗病患和阻止传播的唯一方法 (中国卫生部 2010)。 虽有疫苗在研发中， 但 是保护效率仍然未知， 且大规模实施接种仍需较长时日。
因此， 本领域需要大力研究能够防治手足口病的药物， 特别是研究抑制 EV71 和
CVA16病毒感染的药物以从源头上进行防治。 发明内容
本发明的目的在于提供一种治疗病毒疾病的成分和方法。
在本发明的第一方面， 提供一种 P2X 受体拮抗剂的用途， 用于制备抑制正义单链 RNA病毒微小核糖核酸病毒的组合物。
在一个优选例中， 所述的 P2X 受体拮抗剂用于制备预防、 缓解或治疗手足口病的 组合物。
在另一优选例中， 所述的组合物还用于： 全身给药或肠胃外给药； 较佳地， 所述的 组合物用于口服、 静脉注射、 肌肉注射或吸入给药。
在本发明的另一方面，提供一种抑制正义单链 RNA病毒微小核糖核酸病毒的方法， 包括： 给予需要抑制病毒的对象有效量的 P2X受体拮抗剂。
在一个优选例中， 所述的对象是病毒感染的哺乳动物， 包括肠道病毒 71 型或柯萨 奇病毒感染的人， 猴子或鼠等， 更佳地是手足口病患者。
在另一优选例中， 所述的 P2X受体拮抗剂选自下组： PPADS , iso-PPADS , PPNDS ,
Suramin, NF023 , TNP-ATP , NF279 , NF157, Evans Blue, 和 /或它们的类似物或 衍生物， 和 /或它们的药学上可接受的盐。
在另一优选例中， 所述的病毒是肠道病毒。
在另一优选例中， 所述的病毒是肠道病毒 A ( enterovirus A) 。
在另一优选例中， 所述的病毒是人肠道病毒 71型和柯萨奇病毒。
更佳地， 所述的病毒是人肠道病毒 71型， C4亚型和柯萨奇病毒 A16亚型。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1、 不同 E02浓度下用 EV71临床分离株感染 RD细胞， M0I为 0.1。 培养 46小 时后提取培养上清中的病毒 RNA, 定量 RT-PCR测定 EV71相对病毒量。 表中 2、 3、 4 列数字代表 3次重复试验的结果， 右图是三次结果的平均值。
图 2、 在不同 E02浓度下用 10倍梯度稀释的 EV71感染 RD细胞， 培养 3-4天后光 学显微镜下观察 CPE (细胞病变现象)， 并用结晶紫染色， 计算半数组织培养感染剂量 (TCID50)。
图 3、 在不同 E02浓度下用 EV71感染 12孔板中培养的 RD细胞， 培养 5〜7天后 染色计算空斑形成单位 (PFU)。
图 4、 在不同 E02浓度下用 10倍梯度稀释的 EV71 M.A.V.感染 RD细胞， 培养 3至 4天后光学显微镜下观察 CPE, 并用结晶紫染色， 计算半数组织培养感染剂量 (TCID5())。
图 5、 在不同 E02浓度下用 EV71 M.A.V.感染 12孔板中培养的 RD细胞， 培养 5〜
7天后染色， 计算空斑形成单位 (PFU)。
图 6、 在不同 E02浓度下用 10倍梯度稀释的柯萨奇病毒 A16感染 RD细胞， 培养 2天后光学显微镜下观察 CPE, 并用结晶紫染色， 计算半数组织培养感染剂量 (TCID5())。 图 7、 用 EV71临床分离株感染三日龄新生 ICR小鼠， 感染后 5天采血分离血清； 提取血清中病毒 RNA, —步法实时定量 RT-PCR检测病毒载量。
图 8、 CVA16在 ICR新生小鼠各器官或组织中的分布。 其中， 1号和 2号表示从分 别的 2只小鼠获取的血液样品。
图 9、 用 CVA16病毒感染三日龄新生 ICR小鼠， 感染后 6天取组织， 采血分离血 清， 提取组织和血清 RNA, 测定 CVA16病毒 RNA, 计算病毒载量。
图 10、 用 Celltiter Glo reagent 测定不同浓度 E02对于 RD细胞的细胞毒性。
图 11、 三日龄 ICR小鼠分别腹腔注射 50微升剂量为 50mg/kg， 20mg/kg的 E02或 者 50微升 DMEM培养基， 连续 4天， 每日注射， 观察毒性的结果。
图 12、 E02加入时间测试， 测试在感染前 (B， C)、 感染中 (D， E)、 感染后 (F)分别 加入 E02， E02抑制 EV71感染的有效性。
图 13、 在不同 PPADS浓度下用 EV71临床分离株感染 RD细胞， 测定 PPADS对 EV71复制的抑制作用。
图 14、 通过 RT-PCR方法， 测定六种 P2X亚型在 RD细胞中的 mRNA表达情况。 图 15、 E02抑制 EV71分离株 SH-TS和 SH-RS在 RD细胞中复制。
图 16、 E02降低 RD细胞中 EV71分离株 SH-TS, SH-RS的 PFU。
图 17、 E02抑制 EV71分离株 SEP-4在 Vera细胞中复制。
图 18、 E02抑制 EV71在恒河猴体内的复制。
图 19、 E02抑制 EV71引起的恒河猴体温的升高。
图 20、 P2X受体拮抗剂抑制 RD细胞中 EV71复制。
图 21、 其它一些拮抗剂和激动剂对于 EV71的作用。 具体实施方式
本发明人经过深入的研究， 首次揭示了一种 P2X受体拮抗剂的新用途， 用于制备 一种病毒的组合物。所述的 P2X受体拮抗剂通过抑制病毒发挥预防或治疗手足口病的作 用。
P2X受体拮抗剂及其用途
P2X是一类 ATP 门控离子通道蛋白家族， 表达于细胞膜， 一般为同源或异源三聚 体， 由一个胞外区、 两个跨膜区和两个胞内区组成。 P2X 家族包括七个亚型。 经胞外 ATP触发， P2X离子通道打开， 引发钙离子内流， 胞内钙离子聚集， 通过 MAPK, PKC 和钙调蛋白激发系列下游信号转导 CErb， Liao et al. 2006)。
本发明人深入研究后发现， 一些 P2X 受体拮抗剂对于抑制病毒是有用的， 所述的 病毒是正义单链 RNA 病毒微小核糖核酸病毒科的病毒， 包括口蹄疫病毒属 ( Aphtho virus ) ， 禽月干炎病毒属 ( Avihepato virus) ， 、病毒属 ( Cardio virus) ， 泰勒病 毒属 ( Theilo virus ) ， 肠病毒属 ( Enterovirus ) ， 马鼻病毒属 ( Erbo virus ) ， 肝病毒属 ( Hepato virus) ， 嵴病毒属 （Kobuvirus) ， Parechovirus 等； 更佳地， 所述的病毒是肠 道病毒属， 包括肠道病毒 A-H类， 鼻病毒 A-C ( Rhino virus A-C) ； 更佳地， 所述的病 毒是肠道病毒 A类 (Enterovirus A), 包括沸沸肠道病毒 ( Baboon enterovirus) ， 人柯萨 奇病毒 A2-8， A10, A12, A14, A16型， 人肠道病毒 71， 76, 89， 90-92型等； 更佳地， 包括肠道病毒 71型 （EV71) (包括 A， B和 C亚型)， 柯萨奇病毒 （包括 A和 B类， 优选 包括 A16亚型； CVA16)。
公知地， 肠道病毒 71型或柯萨奇病毒 A16亚型感染是手足口病的主要病因。 中国 卫生部公布的 《手足口病诊疗指南 (2010年版)》 及多项已发表的研究都指出， EV71 和 CVA16是主要病原。 因此， P2X受体拮抗剂可以用于防治手足口病。
作为本发明的一种选择方式， 所述的 P2X受体是 P2X1-7亚型受体。
作为本发明的另一优选方式， 所述的 P2X受体拮抗剂选自： PPADS , Iso-PPADS , PPNDS, Suramin, NF023(Suramin衍生物 TNP-ATP, NF279, NF 157， Evans Blue。
作为本发明的更优选方式， 所述的 P2X 受体拮抗剂可以是： Suramin, PPADS , iso-PPADS, PPNDS , NF023 , NF279, TNP-ATP, NF157, Evans Blue。
本发明还包括上述所列举的各种化合物的异构体、 外消旋体、 药学上可接受的盐、 水合物、 前体、 衍生物或类似物， 只要所述的异构体、 外消旋体、 药学上可接受的盐、 水合物、衍生物或类似物也具有抑制病毒 （如肠道病毒 71型或柯萨奇病毒 A16亚型） 的 功能。
所述的 "异构体"包括： 构象异构体， 光学异构体 (如对映异构体和非对映异构体)， 几何异构体 (如顺反异构体)。
所述的 "衍生物或类似物" 是指具有上述所列举的 P2X受体拮抗剂类似的结构式， 特别是具有相同的母核结构的， 但一些化合物基团被相近的基团所取代的化合物， 该化 合物仍然保留有抑制病毒 （如肠道病毒 71型或柯萨奇病毒 A16亚型） 的功能。 例如， H 被 C1-C4烷基取代； C1烷基被 C2烷基取代； 卤素基团 (包括 F、 Cl、 Br、 I)之间发生取 代； OH、 OCH3、 OCH2CH3、 OCH2CH3之间发生取代等。
所述的 "药学上可接受的盐" 是指上述化合物与无机酸、 有机酸、 碱金属或碱土金 属等反应生成的盐。 这些盐包括 (但不限于)： （1)与如下无机酸形成的盐： 如盐酸、 氢溴 酸、 氢碘酸、 硫酸、 硝酸、 磷酸； （2)与如下有机酸形成的盐， 如乙酸、 乳酸、 柠檬酸、 琥珀酸、 延胡索酸、 葡萄糖酸、 安息香酸、 甲烷磺酸、 乙烷磺酸、 苯磺酸、 对甲苯磺酸、 草酸、 丁二酸、 酒石酸、 马来酸、 或精氨酸。 其它的盐包括与碱金属或碱土金属 (如钠、 钾、 钙或镁)形成的盐， 铵盐或水溶性的胺盐 (如 N-甲基葡糖胺盐)、低级的烷醇铵盐以及 其它药学上可接受的胺盐 (比如甲胺盐、 乙胺盐、 丙胺盐、 二甲基胺盐、 三甲基胺盐、 二 乙基胺盐、 三乙基胺盐、 叔丁基胺盐、 乙二胺盐、 羟乙胺盐、 二羟乙胺盐、 三羟乙胺盐， 以及分别由吗啉、 哌嗪、 赖氨酸形成的胺盐)， 或其它常规的 "前体药物" 的形式。 化合 物具有一个或多个不对称中心。 所以， 这些化合物可以作为外消旋的混合物、 单独的对 映异构体、 单独的非对映异构体、 非对映异构体混合物、 顺式或反式异构体存在。
所述的 "化合物的前体" 指当用适当的方法服用后， 该化合物的前体在病人体内进 行代谢或化学反应而转变成本发明前面所列举的 P2X受体拮抗剂化合物， 或 P2X受体 拮抗剂化合物所组成的盐或溶液。 化合物的前体包括但不局限于所述化合物的羧酸酯、 碳酸酯、 磷酸酯、 硝酸酯、 硫酸酯、 砜酯、 亚砜酯、 氨基化合物、 氨基甲酸盐、 偶氮化 合物、 磷酰胺、 葡萄糖苷、 醚、 乙縮醛等形式。 筛药方法
本发明人的研究发现一类 P2X受体拮抗剂能抑制病毒的复制，该抑制作用可能是通 过靶向 P2X受体发挥的。
在得知了 P2X受体拮抗剂通过选择性地结合于 P2X受体， 发挥抑制病毒的作用后， 可以基于该特征来筛选结合于 P2X受体的物质。之后， 可从所述的物质中找到对于抑制 病毒真正有用的药物。
因此， 本发明提供筛选防治手足口病的潜在药物的方法， 所述方法包括： （1) 提供 一包含 （如表达） P2X受体的体系； （2) 将候选物质给予步骤 (1)的体系，观察候选物质与 P2X受体的结合情况； 若候选物质能够与 P2X受体结合， 则该候选物质是防治手足口病 的潜在药物。 所述的包含 （如表达） P2X受体的体系例如可以是细胞体系， 所述的细胞 可以是内源性表达 P2X受体的细胞； 或可以是重组表达 P2X受体的细胞。 所述的包含 P2X受体的体系还可以是亚细胞体系、 溶液体系、 组织体系、 器官体系或动物体系 （如 动物模型， 优选非人哺乳动物的动物模型， 如鼠、 兔、 羊、 猴等） 等。 检测一个化合物 是否与受体或受体的部分亚基结合是本领域已知的技术。
在本发明的优选方式中， 在进行筛选时， 为了更易于观察到 P2X受体与候选物质结 合情况的差异， 还可设置对照组， 所述的对照组可以是不添加所述候选物质的包含 P2X 受体的体系。
所述的候选物质可以包括 （但不限于)： 小分子化合物， 多肽， 配体。 较佳地， 所述 的候选物质是小分子化合物。 例如， 所述的候选物质可以是各种 P2X受体拮抗剂。
作为本发明的优选方式， 所述的方法还包括： 对获得的潜在物质进行进一步的细胞 实验和 /或动物试验， 以进一步选择和确定对于抑制病毒真正有用的物质。
另一方面， 本发明还包括采用所述筛选方法获得的抑制病毒的物质。 组合物
本发明的化合物可以制备药物组合物， 药用载体可以是固体或液体， 固体制剂包括 散剂、 片剂、 丸剂、 胶囊、 扁囊剂、 栓剂和可分散颗粒剂， 固体载体可以是一种或多种 物质， 它们可以作为稀释剂、 矫味剂、 黏合剂、 防腐剂、 片剂崩解剂或包囊材料。 在散 剂中， 载体是微细分的固体， 本发明的化合物与微细分的活性组份存在于混合物中。 在 片剂中， 此化合物与所需的粘合载体以适当比例混合， 并压制成所需的形状和大小。 适 宜的载体是碳酸镁、 硬脂酸镁、 滑石、 糖、 乳糖、 果胶、 糊精、 淀粉、 明胶、 羧甲基纤 维素、 羧甲基纤维素钠、 低熔点腊和可可脂等。 同样， 扁囊剂或锭剂、 片剂、 散剂、 胶 囊、 丸剂、 扁囊剂和锭剂可以是适合于口服给药的固体剂型。
溶液形式的制剂包括溶液、 悬浮剂和乳剂， 例如水或含水丙二醇溶液。 对于非肠道 注射液体制剂， 可以在含水聚乙二醇溶液中制备。 适于口服的含水溶液可以通过将活性 组份溶解于水中， 并按照需要加入适宜的着色剂、 矫味剂、 乳化剂和增稠剂制备。
所述的组合物可用于全身给药、 局部给药或肠胃外给药等。
适于口服的含水混悬剂可以通过将微细分的活性组份分散于含水粘性物质中制备， 如天然或合成胶、 树脂、 甲基纤维素、 羟甲基纤维素钠及其他熟知的混悬剂。
在治疗用途中， 用于本发明的化合物以起始剂量每天 0.01 mg至 200 mg/kg体重， 更佳地 0.25 mg至 100 mg/kg体重， 更佳地 0.5mg至 50mg/kg体重。 但是， 这些剂量可 以根据患者的需要、 被治疗病症的严重性以及使用的化合物而变化， 一般来说， 开始以 小于该化合物最佳剂量的较小剂量治疗， 此后， 小量增加此剂量达到最佳效果， 方便起 见， 如果需要可将总日剂量再细分为一天内分次给药。
本发明的药物组合物还可同时与其它治疗剂或辅剂联合使用。 试剂盒
本发明所述的 P2X受体拮抗剂或含有 P2X受体拮抗剂的组合物， 可以被置于试剂 盒中， 以便于出售或使用。
所述的 P2X受体拮抗剂或含有 P2X受体拮抗剂的组合物可以被制成单元剂型的形 式， 置于试剂盒中。 "单元剂型"是指为了服用方便， 将本发明的 P2X受体拮抗剂或含 有 P2X受体拮抗剂的组合物制备成单次服用所需的剂型， 包括但不限于各种固体剂 (如 片剂)、 液体剂、 胶囊剂、 缓释剂。 当制备成单元剂型时， 可每天服用所述单元剂型的组 合物 1-3剂。
所述的试剂盒中还包括：将 P2X受体拮抗剂给予需要抑制病毒的对象 （如患有病毒 疾病的患者， 或含有病毒的场所） 的给药说明书， 以知道人们正确地用药。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002中所述的条件， 或 按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份数按体积计算。 I. 实验材料 1、 细胞系
横纹肌肉瘤细胞 （RD 细胞） 购自 ATCC , 编号 VR-805。 细胞培养基为在含有 10%(v/v)FBS的 DMEM, 加入 l%(w/v)氨苄青霉素 /链霉素溶液。 2、 病毒株
EV71临床分离株： EV71 FY 573， 由 2008年中国安徽省手足口病例样本中分离得 至 lj， 基因型为 EV71 C4, Genbank登录号 HM_064456。 由中国科学院上海生命科学研究 院提供。
EV71临床分离株 SH-TS和 SH-RS , 由中国科学院上海生命科学研究院提供， 基因 型为 C4。
EV71临床分离株 SEP-4, 由柬埔寨巴斯德研究所 （Institut Pasteur of Cambodia)从 柬埔寨临床手足口病样本分离所得并提供， 基因型为 C4。
EV71-FY23 株： 由中国医学科学院医学生物学研究所病毒免疫室提供， 批号： 20121001, Genebank 登录号 EU812515.1。
EV71 M.A.V. : EV71小鼠适应株， 将 EV71 FY 573在新生小鼠内反复传代获得。
CVA16: 柯萨奇病毒 A16亚型， shzh05-l (GenBank# EU262658)。
3、 化合物
E02购自 Sigma- Aldrich, 以 Suramin Sodium给药。
猴子实验所用 suramin由 Bayer公司提供， 以 Suramin自由酸给药。
iso-PPADS(0683-10mg), NF 279 (1199-lOmg), PPNDS(1309)购自 Tocris。
除非另外说明， 其他化合物均购自 Sigma-Aldrich和 Tocis。
4、 RT-PCR引物及探针 (5，〜3，）：
EV71弓 I物， 正向： CCCTGAATGCGGCTAATC (SEQ ID NO: 1);
EV71引物， 反向： ATTGTC ACC ATAAGC AGCC A (SEQ ID NO: 2)；
EV71探针： FAM 6-羧基荧光素) -AACCGACTACTTTGGGTGTCC GTGTTTC (SEQ ID NO: 3)-TAMRA (6-羧基四甲基罗丹明）；
GAPDH正向弓 I物： GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 4)；
GAPDH反向引物： GAAGATGGTGATGGGATTTC (SEQ ID NO: 5)；
P2X1-164F: CCTCTTCGAGTATGACACC (SEQ ID NO: 6)；
P2X1-164R: CAGAGACACTGCTGATGAG (SEQ ID NO: 7)；
P2X2-202F: CTGGACATGCTGGGAAACG (SEQ ID NO: 8)；
P2X2-202R: TGCCCTTGGAGAAGTGGAAT (SEQ ID NO: 9)；
P2X3-153F: GGCTCGACAGCGTTTCT (SEQ ID NO: 10)； P2X3-153R: TGCCAGCATTCCCGTAT (SEQ ID NO: 11)；
P2X4-201F: TGGGATGTGGCGGATTAT (SEQ ID NO: 12)；
P2X4-201R: TACGCACCTGCCTGTTGAGA (SEQ ID NO: 13)；
P2X5-220F: TCTTTGCCTGGTGCCCGTTG (SEQ ID NO: 14)；
P2X5-220R: ATCACGGAGCCCAGTCGGAAG (SEQ ID NO: 15)；
P2X6-205F: GGAGGACAAAGTATGAGGAGG (SEQ ID NO: 16)；
P2X6-205R: GAATGGGTTGGCAAGTGG (SEQ ID NO: 17)；
P2X7-175F: CGTGGAGAAGTGAAGAAG (SEQ ID NO: 18)；
P2X7-175R: TCGGTCAGAGGAACAGAGC (SEQ ID NO: 19)。
5、 试剂及试剂盒
DMEM: Invitrogen cat# 11965118;
FBS: Invitrogen cat# 10099141 ;
TRYPSIN 0.25% EDTA: Invitrogen cat# 525200072;
病毒 RNA提取试剂盒： QIAamp Viral RNA Mini Kit, QIAGEN cat#52906;
细胞总 RNA提取试剂盒： RNeasy Mini Kit (250)， QIAGEN cat#74106;
组织保存液： RNAstore样本保存液， 天根生化科技 (北京)有限公司， DP408-02; 组织 RNA提取试剂盒： RNAprep pure动物组织总 RNA提取试剂盒， 天根生化科技 (北京)有限公司， DP43 ;
Taqman real time RT-PCR reagent： ABI taqMan® One-Step RT-PCR, ABI cat#
4309169；
SYBR real time RT-PCR reagent: QIAGEN QuantiTect SYBR Green RT-PCR Kit (200), Cat# 204243；
一步法 RT-PCR试剂： QIAGEN OneStep RT-PCR Kit (100); Cat# 201212；
DNA 电泳琼脂糖： Biowest Argarose, Cat# BW-R0100;
DNA marker; DL2,000 DNA Marker, Cat# D501 ;
Crystal violet: Santa Cruz cat# sc-207460;
Seaplaque Agarose: lonza/amaxa 4 50101；
CellTiter Glo: Promega G7572;
PENICILLIN STREPTOMYCIN: Invitrogen Cat# 15140122
6、 缩写及简称
CPE: cytopathic effect, 细胞病变作用；
CT: cycle threshold, 循环阈值；
EC50: 50% effective concentration, 半数有效剂 EV71 : Enterovirus 71 , 肠道病毒 71型;
Evans Blue ： 6，6-[(3，3 '-Dimethyl[l，l '-biphenyl]-4，4'-diyl)bis(azo)bis[4-amino- 5-hydroxy- 1 ,3-naphthalenedisulphonic acid] tetrasodium salt;
HFMD: Hand Foot and Mouth Disease, 手足口病；
iso-PPADS: pyridoxal-phosphate-6-azophenyl-2' , 5 '-disul honic acid;
M.A.V. : Mouse Adapted Virus, 小鼠适应病毒；
MAPK: Mitogen-activated protein kinases, 促分裂素原活化蛋白激酶；
MOI: Multiplicity of infection, 感染复数；
NF 023 ： 8,8'-[carbonyl¾z'5(imino-3,l-phenylenecarbonylimino)]¾z5-l,3,5- naphthalene-trisulphonic acid, hexasodium salt;
NF 157 ： 8 , 8 ' - [ C arbo ny lb is [ imino - 3 , 1 -p heny lenec arbony limino (4 - f uoro -3,1- phenylene)carbonylimino]]；
NF 279 ： 8,8 '-(carbonylbis(imino-4 ， 1 -phenylenecarbonylimino-4, 1 - phenylenecarbonylimino)) bis(l,3,5-naphthalenetrisul fonic acid)
PFU: plaque forming unit
PKC: Protease kinase C, 蛋白激酶 C;
PPADS: pyridoxal-phosphate-6-azophenyl-2' , 4'-disul honic acid; 口多磷酸盐 - 6—苯 偶氮 -2'， 4'-二磺酸；
PPNDS: Pyridoxal-5 ' -phosphate-6-(2 ' -naphthylazo-6 ' -nitro-4 ' ,8 ' -disulfonate) tetrasodium salt, 哆 -5 '-磷酸 -6-(2'-naphthylazo-6'-硝基 -4'， 8'-磺酸钠）四钠盐；
RD cell: Rhabdomyosarcoma cell, 横纹肌肉瘤细胞；
T.O.A. : Time of Addition assay, 加入时间实验；
TCID50: 50% Tissue Culture Infective dose, 半数组织培养感染剂量
TNP-ATP: trinitrophenol-ATP, 苦味酸 -ATP;
UTP: uridine triphosphate, 尿苷三磷酸；
7、 化合物结构式及 CAS号
化合物 CAS注册号或化学式
CAS: 129-46-4
Figure imgf000010_0001
Figure imgf000011_0001
Evans Blue 314-13-6
Figure imgf000012_0001
II. 实施例
EV71 和 CVA16 是导致手足口病的主要病原， 本发明人在以下实施例中测定了 E02(Suramin Sodium)或其类似物抑制 EV71和 CVA16体外感染及体内感染的能力。 实施例 1、 E02抑制病毒 EV71临床分离株（EV71 FY 573)感染 RD细胞（体外试验) 1、 E02能够抑制 EV71在 RD细胞中的复制
在不同 E02 浓度下用 EV71 临床分离株 （EV71 FY 573 ) 感染 RD 细胞， MOI (multiplicity of infection感染复数） 为 0.1。 培养 46小时后提取培养上清中的病毒 RNA, 定量 RT-PCR测定 EV71相对病毒量。 详细步骤如下：
①细胞接种： 感染前 24小时， 接种 RD细胞于 96孔细胞培养板， 每孔 5 X 104个 细胞；
②感染：
a.药物预孵育病毒： 用 DMEM稀释病毒储存液至 5 X 104 TCID50/ml, 每孔 100微 升分装至 96孔板， 并加入 11微升相应浓度溶解于 DMEM的 E02或者 DMEM培养基，
37°C孵育 1小时；
b.药物预孵育细胞： 弃去 96孔板中的细胞培养液， 每孔加入 100微升 DMEM, 再 加入 11微升相应浓度的 E02溶液或者 DMEM培养基， 37°C孵育 1小时；
c孵育 1小时后， 弃去细胞培养板中的药物， 将 111微升药物预孵育的病毒转移至 细胞培养板， 37°C孵育 1小时；
d.孵育后， 弃去上清， 50微升 DMEM洗细胞两次， 每孔加入 180微升含 2% FBS 的 DMEM培养基， 20微升相应浓度的 E02 DMEM溶液或者 DMEM培养基， 将细胞培 养板置于 37°C， 5%(v/v)二氧化碳的培养箱培养；
③病毒收获和 RNA提取： 被感染的细胞培养 46小时后， 转移 140微升上清至 96 孔深孔板，用病毒 RNA提取试剂盒 （QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906) 提取病毒 RNA， 按照试剂盒标准操作程序进行；
④病毒载量测定： 用 ABI Taqman一步法 RT-PCR试剂盒 （ABI TaqMan® One-Step RT-PCR, cat# 4309169) 在 ABI 7900HT 384孔板 PCR系统检测 EV71 5'UTR基因。 反 应体系和 PCR程序见表 1和表 2。 EV71 5 'UTR定量 RT-PCR热循环程序 (Taqman);
⑤将 PCR的 CT值用 PCR标准曲线转化为病毒载量 （标准曲线由已知滴度的病毒 十倍梯度稀释， 抽提 RNA， RT-PCR测定 CT值得到）。 表 1、 EV71 5 'UTR实时定量 RT-PCR反应体系（Taqman)
Figure imgf000013_0001
表 2、 EV71 5 ' UTR 定量 RT-PCR热循环程序 QTaqman)
Figure imgf000013_0002
结果显示， E02能够抑制 EV71在 RD细胞中的复制， EC50(50%有效浓度)为 6.93 微摩， 3.44微摩可降低 EV71复制 10倍； 5.19微摩降低 EV71复制 100倍； 6.45微摩降 低 1000倍； 7.63微摩降低 10000倍， 详见图 1。
2、 E02显著降低 RD细胞中 EV71 TCID50
在不同 E02浓度下用 10倍梯度稀释的 EV71 (临床分离株 EV71 FY 573 ) 感染 RD 细胞， 培养 3-4天后光学显微镜下观察 CPE (细胞病变现象)， 并用结晶紫染色， 计算半 数组织培养感染剂量。 详细步骤如下：
①细胞接种： 感染前 24小时， 接种 RD细胞于 96孔细胞培养板， 每孔 5 X 104个 细胞；
②感染：
a. 药物预孵育病毒： 用 DMEM培养基 10倍梯度稀释病毒储存液， 每孔 100微升分 装至 96孔板， 并加入 1 1微升相应浓度溶解于 DMEM的 E02， 37 °C孵育 1小时；
b. 药物预孵育细胞： 弃去 96孔板中的细胞培养液， 每孔加入 100微升 DMEM, 再 加入 1 1微升相应浓度的 E02溶液， 37°C孵育 1小时；
c 孵育 1小时后， 弃去细胞培养板中的药物， 将 1 1 1微升药物预孵育的病毒转移至 细胞培养板， 37°C孵育 1小时；
d. 孵育后， 弃去病毒药物混合液， 50微升 DMEM洗细胞两次， 每孔加入 180微升 含 2% FBS的 DMEM培养基， 20微升相应浓度的 E02 DMEM溶液， 将细胞培养板置于 37°C , 5%二氧化碳的培养箱培养；
③培养 3至 4天后， 光学显微镜下观察 CPE, 并用结晶紫染色， 用 Reed-Muench 算法计算半数组织培养感染剂量 TCID50 ;
结果显示， E02能够抑制 RD细胞中 EV71病毒感染导致的 CPE， E02降低 RD细胞 中 EV71 TCIDso的半数有效剂量为 4.7微摩， 见图 2。 因此， E02显著降低 RD细胞中 EV71 TCID50。
3、 E02降低 RD细胞中 EV71空斑形成单位
在不同 E02浓度下用 EV71 (临床分离株 EV71 FY 573 ) 感染 12孔板中培养的 RD 细胞， 培养 5〜7天后染色计算空斑形成单位。 详细步骤如下：
①细胞接种： 感染前 24小时， 接种 RD细胞于 12孔板， 每孔 2 X 105个细胞；
②感染细胞：
a. 药物预孵育病毒： 用 DMEM培养基 10倍梯度稀释病毒储存液至 50000PFU/ml，
5000PFU/ml和 500PFU/ml， 每孔 500微升分装至 12孔板， 并加入 56微升相应浓度溶解 于 DMEM的 E02， 37°C孵育 1小时；
b. 药物预孵育细胞： 弃去 12孔板中的细胞培养液， 每孔加入 500微升 DMEM, 再 加入 56微升相应浓度的 E02溶液， 37°C孵育 1小时；
c 孵育 1小时后， 弃去细胞培养板中的药物， 将 556微升药物预孵育的病毒转移至 细胞培养板， 37°C孵育 1小时；
d. 孵育后， 弃去病毒药物混合液， 300微升 DMEM洗细胞两次， 每孔加入 150微 升相应浓度的 E02 DMEM溶液，再加入 1350微升预热熔解 G令却至 37〜40°C)的含有 1% 低熔点琼脂糖 seaplaque, 2%FBS的 DMEM培养基。 将细胞培养板置于 37°C， 5%二氧 化碳的培养箱培养；
③培养 5-7天后， 移去凝胶， 用结晶紫染色， PBS冲洗一次， 计空斑数并计算空斑 形成单位。
结果显示， E02能够抑制 EV71在 RD细胞中形成的空斑数量。 E02降低 RD细胞中 EV71 PFU的半数有效剂量为 2.6微摩， 见图 3。 因此， E02可以降低 RD细胞中 EV71 空斑形成单位。
综上， 通过实时定量 RT-PCR, TCID5Q和 PFU三种测试方法， 结果均表明 E02能够 抑制 EV71临床分离株感染 RD细胞： 抑制 EV71在 RD细胞中复制的 EC50为 6.93微 摩； 降低 RD细胞中 TCID5。的 EC50为 4.7微摩； 降低 PFU的 EC50为 2.6微摩。 实施例 2、 E02抑制肠道病毒 EV71鼠适应株感染 RD细胞 （体外试验) 1、 E02降低 RD细胞中 EV71 M.A.V. TCID50
在不同 E02浓度下用 10倍梯度稀释的 EV71 M.A.V.感染 RD细胞， 培养 3至 4天 后光学显微镜下观察 CPE, 并用结晶紫染色， 计算半数组织培养感染剂量。 具体步骤同 实施例 1中相应的 TCID5Q测试步骤。
结果显示， E02降低 RD细胞中 EV71 M.A.V. TCID50的半数有效剂量为 8.07微摩， 见图 4。 因此， E02降低 RD细胞中 EV71 M.A.V. TCID50。
2、 E02降低 RD细胞中 EV71 M.A.V. PFU
在不同 E02浓度下用 EV71 M.A.V.感染 12孔板中培养的 RD细胞，培养 5〜7天后 染色， 计算空斑形成单位。 详细步骤同实施例 1中相应的 PFU测试方法。
E02降低 RD细胞中 EV71 M.A.V. PFU的半数有效剂量为 2.0微摩， 见图 5。 因此，
E02降低 RD细胞中 EV71 M.A.V. PFU。 综上， TCID50和 PFU测试表明， E02能够抑制小鼠适应 EV71病毒株感染 RD细 胞， EC50分别为 8.07微摩和 2.0微摩。 实施例 3、 E02抑制柯萨奇病毒 A16(CVA16)感染 RD细胞 （体外试验）
在不同 E02浓度下用 10倍梯度稀释的柯萨奇病毒 A16感染 RD细胞， 培养 2天后 光学显微镜下观察 CPE, 并用结晶紫染色， 计算半数组织培养感染剂量。 具体步骤同实 施例 1中 TCID5Q测试方法。
结果显示， 25微摩 E02使 RD细胞中 CVA16滴度由的 1 X 109 TCID ml降至 I X
104 TCID50/ml， 见图 6。 因此， E02降低 RD细胞中 CVA16 TCID50. 以上实施例 1-3的测定结果表明， E02能够在体外抑制 EV71和 CVA16感染 RD细 胞。 E02抑制 EV71临床分离株的 EC50小于 6.93微摩， 10微摩 E02能够完全抑制 E02 临床株和鼠适应株感染 RD细胞， 并使 CVA16在 RD细胞中的 TCID5Q降低。 实施例 4、 E02抑制肠道病毒 EV71感染 ICR新生小鼠
用 EV71临床分离株（EV71 FY 573 )感染三日龄新生 ICR小鼠， 测试小鼠体内 E02 抑制 EV71的能力。 实验小鼠分为三组： 药物组、 感染组、 安慰组。 小鼠二日龄时， 药 物组腹腔注射 50微升 50mg/kg剂量 E02， 感染组和安慰组腹腔注射 50微升 DMEM培 养基； 三日龄时药物组腹腔注射 50mg/kg剂量 E02和 5 X 107 TCID50 剂量的 EV71临床 分离株病毒混合物 (50微升），感染组注射 5 X 107 TCID5Q病毒 (25微升)和 25微升 DMEM 混合物， 安慰组腹腔注射 50微升 DMEM培养基。
感染后 5天采血分离血清。 提取血清中病毒 RNA(病毒 RNA提取试剂盒， QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906); 用一步法实时定量 RT-PCR试剂盒 (QIAGEN QuantiTect SYBR Green RT-PCR Kit)在 ABI 7900HT 384孔板 PCR系统检测 EV71 5 'UTR 基因及 GAPDH基因拷贝， 反应体系和 PCR程序见表 3和表 4。
表 3、 EV71 5 'UTR和 GAPDH基因实时定量 RT-PCR反应体系（SYBR)
Figure imgf000016_0001
表 4、 EV71 5 'UTR和 GAPDH基因实时定量 RT-PCR热循环程序 (SYBR)
Figure imgf000016_0002
计算 EV71和 GAPDH RNA载量比值， 感染组平均值为 15.0， 药物组平均值为 1.8， 见图 7。 结果表明， E02降低小鼠血清中 EV71病毒量， 50mg/kg的 E02能够降低感染 EV71的 ICR新生小鼠血清中的病毒量。 实施例 5、 E02抑制柯萨奇病毒 A16感染 ICR新生小鼠
1、 CVA16在小鼠体内的组织分布
注射 5 X 106 TCIDso CVA16病毒 (50微升)感染三日龄 ICR新生小鼠两只， 感染后 5 天采集血清及组织 (脑， 骨髓， 心脏， 小肠， 肾， 肝脏， 肺， 肌肉， 脾， 胸腺)。 提取血 清中病毒 RNA (病毒 RNA提取试剂盒， QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906) o组织置于 RNAstore样本保存液 (；天根， DP408-02)保存在 -80°C冰箱，用组织 RNA 提取试剂盒 (RNAprep pure动物组织总 RNA提取试剂盒)提取组织总 RNA。 用一步法实 时定量 RT-PCR试剂盒 (QIAGEN QuantiTect SYBR Green RT-PCR Kit)在 ABI 7900HT 384孔板 PCR系统检测 CVA16病毒基因组及 GAPDH基因拷贝， 引物同 EV71 5 'UTR 引物， 反应体系和 PCR程序见表 3和表 4。 各组织及血清中相对病毒量见图 8。 可见， CVA16病毒主要检出于脑， 脊髓， 肌肉和血清中。
2、 E02抑制 CVA16感染新生 ICR小鼠
用 CVA16病毒感染三日龄新生 ICR小鼠， 测试小鼠体内 E02抑制 EV71 的能力。 实验小鼠分为三组： 药物组， 感染组和安慰组。 小鼠二日龄时， 药物组腹腔注射 50 微 升 50mg/kg剂量 E02， 感染组和安慰组腹腔注射 50微升 DMEM培养基； 三日龄时药物 组腹腔注射 50mg/kg剂量 E02和 1 X 104 TCID50 剂量的 CVA16病毒 (50微升）， 感染组 注射 1 X 104 TCIDso 剂量的 CVA16病毒 (25微升)和 25微升 DMEM混合物， 安慰组腹 腔注射 50微升 DMEM培养基。 感染后 6天取组织， 采血分离血清， 提取组织和血清 RNA, 测定 CVA16病毒 RNA， 处理步骤同前述 " 1 " 。
结果见图 9。 结果显示， 50 mg/kg E02使 ICR新生小鼠的脑， 骨髓， 肌肉和血清中 的 CVA16病毒 RNA均有显著下降。 综上实施例 4-5， 新生 ICR小鼠中的测试表明， 50mg/kg E02可在体内抑制肠道病 毒 EV71和柯萨奇病毒 A16。 实施例 6、 E02安全性、 耐受性及毒性测试
1、 E02无体外细胞毒性
用 Celltitr Glo reagent 测定不同浓度 E02对于 RD细胞的细胞毒性， 如下：
(1) 细胞接种： 测试前 24小时， 每孔 5 X 104个细胞接种 RD细胞于 96孔细胞培养 板；
(2) E02培养细胞：弃去细胞培养板内的培养基，加入 180微升含 2% FBS的 DMEM 培养基， 再加入 20微升相应浓度的 E02 DMEM溶液， 置 37°C， 5% C02 培养箱培养； (3) 培养 46小时后， 用 PROMEGA Celltitr Glo reagent测定细胞 ATP水平， 按照试 剂说明书测定。
测定结果显示： 1 X 10—6至 1 X 104微摩的 E02对 RD细胞均无细胞毒性， 见图 10。
2、 E02对 ICR新生小鼠无毒性
三日龄 ICR小鼠分别腹腔注射 50微升剂量为 50mg/kg， 20mg/kg的 E02或者 50微 升 DMEM培养基， 连续 4天， 每日注射， 观察未见毒性， 见图 11。 实施例 7、 E02抑制肠道病毒 EV71临床分离株 SH-TS和 SH-RS感染 RD细胞 1、 E02抑制 EV71 SH-TS和 SH-RS在 RD细胞中复制
在不同 E02浓度下用 EV71 SH-TS或 SH-RS毒株感染 RD细胞， MOI 0.1。 培养 46 小时后提取培养上清中的病毒 RNA，定量 RT-PCR测定 EV71相对病毒量。定量 RT-PCR 具体步骤参见实施例 1。
结果显示， E02能够抑制 EV71临床分离株 SH-TS和 SH-RS在 RD细胞中的复制， 对于 SH-TS, 7.14微摩可使 EV71复制降低 10倍， 25.35微摩可使 EV71复制降低 100 倍； 对于 SH-RS, 4.41微摩可使 EV71复制降低 10倍； 16.29微摩可使 EV71复制降低 1000倍， 如图 15。
因此， E02抑制 EV71 SH-TS和 SH-RS在 RD细胞中复制。
2、 E02抑制 EV71 SH-TS和 SH-RS感染 RD细胞 (空斑形成单位， PFU)
在不同 E02浓度下用 EV71 SH-TS或者 SH-RS毒株感染 12孔板中培养的 RD细胞， 培养 5〜7天后染色计算空斑形成单位。 详细步骤同实施例 1中 " 3、 E02降低 RD细胞 中 EV71空斑形成单位" 。
结果， E02降低 RD细胞中 EV71 SH-TS PFU的半数有效剂量为 2.24微摩，对于 SH-RS 为 3.47微摩， 如图 16。
病毒复制和 PFU测试表明 E02能够抑制 EV71临床分离株 SH-TS和 SH-RS感染 RD 细胞。 实施例 8、 E02抑制肠道病毒 EV71柬埔寨分离株 SEP-4感染 Vero细胞
在不同 E02浓度下用 EV71 SEP-4病毒株（由 Institut Pasteur of Cambodia提供， Cambodia毒株)感染 Vero细胞， MOI 0.1。 培养 46小时后提取培养上清中的病毒 RNA， 定量 RT-PCR测定 EV71相对病毒量。 具体步骤参见实施例 1。
结果显示， E02能够抑制 EV71 SEP-4株在 Vero细胞中的复制， 5.89微摩 E02可使 EV71复制降低 10倍， 15.49微摩使 EV71复制降低 100倍， 如图 17。 实施例 9、 E02抑制肠道病毒 EV71在恒河猴体内的复制
将已检测 EV71 抗体阴性的 10 只成年恒河猴随机分为 2 组 (恒河猴 (Macaca), 来 源： 中国医学科学院医学生物学研究所， 实验动物使用许可证号： SYXK (滇) 2010-0009， 发证单位： 云南省科学技术厅)， 即： 5只对照组 (年龄约 4岁， 体重约 5.3kg， 注射生理 盐水)， 5只给药组 (；年龄约 4.6岁， 体重约 5.44kg， 注射 50mg/kg suramin)。 采用静脉多 次注射 suramin (50mg/kg)，并于首次注射 suramin的 1天后感染 4.5 lgCCID50EV71 FY-23 病毒 (EV71-FY23 株（由中国医学科学院医学生物学研究所病毒免疫室提供）， 批号： 20121001 , 含量： 7.5 lgCCID50/ml, 保存条件： -80°C 逐日检测病毒在感染成年恒河 猴(>3岁)体内的病毒载量， 观察感染动物的临床症状、体温， 从而分析该药对 EV71 感 染的治疗预防作用。 具体的实验操作流程如下：
1) 感染前一天： 采集本底全血及血清， 测量体温， 观察动物的外观体征、行为活动、 精神状态、 给药局部是否异常等。 并按剂量静脉给药 (50mg/kg suramin 或生理盐水)。
2) 感染当天 (0 天)： 测量体温， 观察动物的外观体征、 行为活动、 精神状态、 给药 局部是否异常等。 并按 4.5 lgCCID50 EV71 FY-23 进行静脉攻毒。
3) 感染期间（1-14天)： 每日测量体温， 观察动物的外观体征、行为活动、 精神状态、 给药局部是否异常等， 检测病毒载量。并于感染后 1、 3、 5天， 按剂量静脉给药 (50mg/kg surami 或生理盐水)。
4) 感染后期 (21和 28天)测量体温， 观察动物的外观体征、 行为活动、 精神状态、 给药局部是否异常等， 检测病毒载量。
血液病毒载量检测
提取全血 RNA: 取 0.2ml, 加入 0.8mlTRNzol-A+， 充分混匀后在室温放置 lOmin; 加入 0.2ml 氯仿， 盖好管盖， 剧烈震荡 15sec， 室温放置 5min， 于 12500rpm， 4°C离 心 20min， 取水相 0.4ml 于另一新管中， 加入等体积异丙醇， 混匀， 室温放置 30 分 钟； 12500rpm， 4°C离心 20min, 弃上清， 加入 1ml 75%乙醇洗涤沉淀， 12500rpm， 4 °C离心 30min.弃上清， 室温放置 30min， 干燥 RNA， 加入 20μ1 RNase-free ddH20, 充分溶解 RNA。 检测病毒载量， 测定方法如下：
1) 标准品稀释：标准品提取（按 TaKaRa MiniBEST Viral RNA/DNA extraction Kit Ver. 3.0 说明操作） ， 取 ΙΟμΙ提取好的标准品， 10倍系列稀释为 106、 105、 104、 103、 102、 101、 10°
2) 引物序列 （Taqman探针法） ：
上游弓 I物 ( ΙΟμΜ) ： 5 '-agcccaaaagaacttcacta-3 '；
下游弓 I物 ( 10μΜ) ： 5 '-atccagtcgatggctgctca-3 '；
探针 5-FAM-agtgatatcctgcagacgggcaccatcc-TAMRA-3。
3) 配制 RT-PCR反应液 （反应液配制在冰上进行） ：
2x One Step RT-PCR Buffer III： 1 Ομΐ
TaKaRa Ex TaqTM HS (51Ι/μ1) ： 0.4μ1
PrimescriptTM RT Enzyme Mix II： 0.4μ1
上游引物 ( 10μΜ) ： 0.4μ1
下游引物 ( 10μΜ) ： 0.4μ1
探针： 0.8μ1
ROX Reference Dye II： 0.4μ1
Total RNA: 2μ1
R ase Free dH20: 5.2μ1
总量： 20μ1
4) 进行 Real Time One Step RT -PCR反应。 1、 病毒载量检测 10只实验动物经 4.5 1gCCID5Q 的 EV71 FY23 株攻击后，对照组动物在攻毒后第 6 和 7天出现一过性的病毒载量升高 (达 1-1.5 X 103拷贝 /ml), 在第 8天后开始下降， 在攻 毒后第 9 天出现一过性的病毒载量升高 (达 5.25 X 102拷贝 /ml), 第 10天开始下降。 给 药组动物分别在攻毒后第 4天、 第 7天和第 13天出现了病毒载量的小幅升高 (平均 1.5 X 101拷贝 /ml), 病毒载量明显低于对照组， 如图 18。
2、 体温检测
对照组动物体温在感染后 1 天就出现一定的上升， 并随之 (约于感染后 3天)降低至 正常范围内； 给药组动物体温基本均在正常范围内， 如图 19。
结论： 在本实验条件下， 静脉途径感染 4.5 lgCCIDso EV71 病毒后， 对照组成年恒 河猴体内病毒载量在特定时间内出现一定的升高，并且体温也出现小幅上升。与之相比， 在该特定时间内给药组成年恒河猴出现病毒载量的小幅升高， 病毒载量明显低于对照 组， 体温检测未出现显著变化。 实施例 10、 E02作用靶点鉴定
1、 E02抑制 EV71进入细胞
用 MOI 10的 EV71临床分离株 EV71 FY573感染 96孔板中的 RD细胞，在感染前， 感染中和感染后三个阶段分别加入 32微摩 E02(溶解于 DMEM)或者 DMEM培养基， 测 定 EV71复制， 确定 E02抑制 EV71感染的作用阶段。 详细步骤如下：
(1) 细胞接种： 感染前 24小时， 接种 RD细胞于 96孔细胞培养板， 每孔 5xl04个 细胞；
(2) 药物预孵育细胞： 弃去 96孔板中的细胞培养液， 每孔加入 100微升 DMEM, 再加入 11微升 320微摩 E02溶液 (药物处理细胞组)或者 11微升 DMEM培养基 (细胞未 经药物处理组)， 37°C孵育 1小时；
(3) 药物预孵育病毒： 用 DMEM稀释病毒储存液至 5 X 106 TCID5Q/ml， 每孔 100微 升分装至 96孔板， 并加入 11微升 320微摩 E02溶液 (Β， C组)或者 11微升 DMEM培 养基 (A， F组）， 37°C孵育 1小时；
(4) 感染： 药物孵育细胞和病毒 1小时后， 弃去细胞培养板中的药物， 将 11 1微升 药物 (B， C组)或 DMEM(A， F组)预孵育的病毒转移至细胞培养板， D，E组移入 100微 升病毒溶液后补加 1 1微升 320微摩 E02， 37°C孵育 1小时；
(5) 病毒感染细胞 1 小时后， 弃去细胞培养板中病毒药物混合液， 50微升 DMEM 洗细胞两次， 每孔加入 180微升含 2% FBS的 DMEM培养基， 20微升 320微摩 E02溶 液 (B， E， F组)或 20微升 DMEM培养基 (A， C， D组）， 将细胞培养板置于 37°C， 5% 二氧化碳的培养箱培养。
(6) 病毒收获和 RNA提取： 被感染的细胞培养 18小时后， 转移 140微升上清至 96 孔深孔板，用病毒 RNA提取试剂盒 （QIAGEN QIAamp Viral RNA Mini Kit, cat # 52906) 提取病毒 RNA， 按照试剂盒标准操作程序进行。
(7) 病毒载量测定：用 ABI Taqman一步法 RT-PCR试剂盒 （ABI taqMan® One-Step RT-PCR, Cat# 4309169) 在 ABI 7900HT 384孔板 PCR系统检测 EV71 5'UTR基因。 反 应体系和 PCR程序见表 1和表 2。
(8) 用 PCR标准曲线将 PCR的 CT值转化为病毒载量 （标准曲线由已知滴度的病毒 梯度稀释， 抽提 RNA， RT-PCR测定 CT值得到）。
高 MOI病毒感染细胞， 可使大部分细胞同步感染， 18小时的培养时间保证病毒只 有一次扩增， 该感染条件可测试药物抑制病毒作用发生在病毒生活周期的哪个阶段 (Bonavia, Franti et al. 2011 ; Daelemans, Pauwels et al. 2011)。 如图 12, E02力口人时间测 试的结果表明在感染前 (B， C)和感染中 (D， E)加入 E02均可抑制 EV71感染， 而感染后 加入 E02(F)， 不能抑制 EV71感染。 若感染过程中已加入药物， 则感染后是否再加入药 物 (D与 E)对病毒复制并无显著影响。 该结果显示， E02通过抑制 EV71病毒进入细胞而 抑制 EV71病毒感染。 2、 P2X受体拮抗剂能够抑制 EV71感染
E02是感知受体 P2X的拮抗剂， 能够抑制 P2X1 , 2， 3和 5亚型 （Khakh, Burnstock et al. 2001 ; Burnstock 2004; Coddou, Yan et al. 2011)受体， 即是 P2X1， 2， 3和 5亚型受 体的拮抗剂。 P2X是一类 ATP门控离子通道蛋白家族， 表达于细胞膜， 一般为同源或异 源三聚体， 由一个胞外区、 两个跨膜区和两个胞内区组成。 P2X家族包括七个亚型。 经 胞外 ATP触发， P2X离子通道打开， 引发钙离子内流， 胞内钙离子聚集， 通过 MAPK, PKC和钙调蛋白激发系列下游信号转导 (Erb， Liao et al. 2006)。 P2X受体在高等动物组 织中广泛分布 CValera, Hussy et al. 1994), 表达于 CNS各部分， 在神经元突触触发， 感 知信号 (如疼痛， 味觉， 听觉等)传入， 平滑肌收縮， 心血管系统血压控制和炎症反应中 都发挥作用（Surprenant and North 2009)。
EV71 感染中枢神经系统会导致急性弛缓性麻痹， 急性传播性脊髓炎和急性横贯性 脊髓炎， 还可引起无菌性脑膜炎和脑炎， 肠病毒感染还可能引起潜在行为和记忆障碍 (Yang, Wang et al. 2009; Rhoades, Tabor-Godwin et al. 2011)。 EV71主要通过诱发病毒 感染患者 CNS炎症， 而导致神经紊乱。 EV71感染可在大脑皮层， 脑干和脊髓各个层次 引起炎症。 EV71感染病人常死于肺水肿或者出血，有研究表明 EV71肺水肿是神经性的 (Solomon, Lewthwaite et al. 2010)。 在死于肺部综合征的 EV71感染病人中， 只有脊髓和 大脑检测到炎症， 而在肺部和心脏检测不到病毒颗粒和炎症。 （Weng， Chen et al. 2010)。 联系 EV71感染引发的疾病的特点， 以及 P2X的分布和生理作用，本发明人认为 P2X在 EV71病理中发挥重要作用。
因此， 本实施例中， 本发明人用化学探针（已知的 P2X抑制剂或激活剂)来确定 P2X 受体在 EV71感染中的作用。 1、 PPADS抑制 EV71感染 RD细胞
PPADS是 E02的类似物（Khakh, Burnstock et al. 2001 ; Burnstock 2004; Coddou, Yan et al. 2011),对 P2X各亚型的抑制作用与 E02—致。在不同 PPADS浓度下用 EV71临床 分离株 FY 573感染 RD细胞， 测定 PPADS对 EV71复制的抑制作用。 详细过程同实施 例 1中" 1 "。并测定各浓度 PPADS对 RD细胞的细胞毒性，详细步骤同实施例 6中" 1 "。
结果见图 13， 64微摩以下浓度 PPADS对 RD细胞无细胞毒性， 抑制 EV71复制的 EC50为 18.9微摩。 2、 P2X受体在 RD细胞中的表达
提取 RD细胞中总 RNA， 一步法 RT-PCR扩增 P2X各亚型 mRNA， 凝胶电泳鉴定 PCR产物长度。 具体步骤如下：
①. 细胞总 RNA提取： 取 2 X 106个 RD细胞， 用 QIAGEN RNeasy试剂盒提取总 RNA, 实验按照试剂盒说明书进行， 产物溶于 50微升无核酸酶水；
②. PCR: —步法 RT-PCR扩增各 P2X亚型和 GAPDH mRNA, 反应体系和循环程序 如表 5和表 6。
③. 电泳： 2%凝胶电泳检测 PCR产物大小。
表 5、 P2X mRNA RT-PCR反应体系
Figure imgf000022_0001
表 6、 P2X mRNA RT-PCR热循环程序
Figure imgf000022_0002
5 72 °C 60秒
3〜5步， 循环 40次
72 °C 10分钟
电泳结果显示， 除 P2X7外， 其它六种 P2X亚型在 RD细胞中均有表达， 结果见图
14。
3、 P2X受体拮抗剂抑制 EV71感染 RD细胞
测定 0.1， 1， 10和 100微摩各 P2X受体拮抗剂及激动剂对 EV71复制的抑制作用， 具体步骤参考实施例 1中 " 1 " ， 但感染后细胞需孵育 3〜4天， 光学显微镜观察并用结 晶紫染色， 确定有无 CPE (细胞病变现象)。 并测定各浓度化合物对 RD细胞的细胞毒性， 在不同浓度化合物中培养 RD细胞后 3〜4天，光学显微镜观察并用结晶紫染色，观察细 胞层有无破坏。 能保护细胞免于感染， 无 CPE且不破坏健康细胞层的化合物， 能够抑制 RD细胞中 EV71的复制。
结果见表 7及图 20，除 E02，PPADS外， P2X受体拮抗剂 iso-PPADS，NF023，NF279， NF157, TNP-ATP, PPNDS , Evans Blue也能够抑制 EV71复制。
表 7、 P2X受体拮抗剂抑制 RD细胞中 EV71复制
Figure imgf000023_0001
对比这些化合物对 P2X和 EV71感染的抑制作用， 如表 8， 表明 P2X受体在 EV71 感染 RD细胞中发挥重要作用。
表 8、 P2X受体与 EV71复制
P2X受体 1 2 3 4 5 6 2/3 抑制 EV71复
Figure imgf000024_0001
注： 激动剂： -: 无活性， + : 激活浓度>1 ΟμΜ, ++： 激活浓度 1〜 ΙΟμΜ, +++： 激活浓度<lμM;
拮抗剂： -: 无活性， +: 拮抗浓度>300nM， ++： 拮抗浓度 = 10〜300ηΜ， +++： ί, 抗浓度 <10ηΜ。 其它一些拮抗剂和激动剂对于 EV71的作用如表 9和图 21。
表 9、 其它一些拮抗剂和激动剂对于 EV71的作用
P2X 受体 EV71
Heteromu
拮抗剂 1 2 3 4 5 6 7 P2Y lOg!o 降低 IC90 ltimer
BBG V V NO NO
MRS 2159 V V NO NO
MRS 2179 V V V V P2Y1 NO NO
NF 1 10 V V V V V NO NO
Ro 0437626 V NO NO
TNP-ATP NO NO
P2X2/3
V V
A-317491 V V NO NO
P2X2/3
V V
AF353 V V NO NO
P2X2/3
RO-3 V V V P2X2/3 NO NO Spinorphin NO NO
5-BDBD V NO NO
A 438079 V V NO NO
A 740003 V V NO NO
A 839977 V V NO NO
AZ 10606120 NO NO
AZ 1 1645373 V V NO NO N-62 V V NO NO oATP V V NO NO
2',5'-ADP P2Y1 NO NO
Heteromu
激动剂 1 2 3 4 5 6 7 P2Y lOg!o 降低 IC90 ltimer
BzATP V V V V V P2Y1 1 NO NO
UTP V NO NO
Heteromu
Modulator 1 2 3 4 5 6 7 P2Y lOg!o 降低 IC90 ltimer
Ivermectin NO NO
GW 791343 NO
Response Heteromu
1 2 3 4 5 6 7 P2Y lOg!o 降低 IC90 Potentiator ltimer
MRS 2219 V NO NO 表中， "空白 " 表示无报道数据， "NO" 表示没有。
对于 Antagonists: V V V 激活浓度<10nM; V V 激活浓度 10nM-300nM， 活浓度 >300nM， 无活性。
对于 Agonists: V V V 激活浓度<l μ M， V V 激活浓度 1-10 μ Μ， V 激活浓度 >10 μ Μ。 因此， Ρ2Χ1〜6亚型在 RD细胞中均有 mRNA表达， 且多个 P2X受体拮抗剂可抑 制 EV71感染 RD细胞复制， 结合 P2X信号转导和 P2X异常与疾病的关系， 提示 P2X 在 EV71感染中发挥重要作用， 且 P2X与 EV71的作用与 EV71感染引起的手足口病的 病理有密切关系。 综上实施例， 可以得出如下结论：
(1) P2X受体拮抗剂化合物 E02能够抑制 EV71病毒感染 RD细胞； (2) 化合物 E02能够降低柯萨奇病毒 A16感染 RD细胞的感染力；
(3) 化合物 E02能够在 ICR新生小鼠体内， 降低血清中 EV71病毒量；
(4) 化合物 E02能够在 ICR新生小鼠体内， 降低脑， 骨髓， 肌肉和血清中 CVA16 病毒量；
(5) E02抑制 EV71在恒河猴体内的复制以及抑制 EV71引起的恒河猴体温的升高。
(6) RD细胞中可检测到 P2X受体 1〜6六种亚型的 mRNA;
(7) 多个 P2X受体拮抗剂能够抑制 EV71感染 RD细胞。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员 可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定 的范围。
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Rhoades, R. E.， J. M. Tabor-Godwin, et al. (201 1). "Enterovirus infections of the central nervous system. " Virology 411(2): 288-305. Solomon, T.， P. Lewthwaite, et al. (2010). "Virology, epidemiology, pathogenesis, and control of enterovirus 71." The Lancet Infectious Diseases 10(11): 778-790.
Surprenant, A. and R. A. North (2009). "Signaling at Purinergic P2X Receptors." Annual Review of Physiology 71(1): 333-359.
Valera, S.， N. Hussy, et al. (1994). "A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP." Nature 371(6497): 516-519.
Weng, K.-F.， L.-L. Chen, et al. (2010). "Neural pathogenesis of enterovirus 71 infection." Microbes and Infection 12(7): 505-510.
Yan, X.-F.， S. Gao， et al. "Epidemic characteristics of hand? foot, and mouth disease in Shanghai from 2009 to 2010: Enterovirus 71 subgenotype C4 as the primary causative agent and a high incidence of mixed infections with coxsackievirus A16." Scandinavian Journal of Infectious Diseases 0(0): 1-9.
Yang, F.， L. Ren, et al. (2009). "Enterovirus 71 Outbreak in the People's Republic of China in 2008." Journal of Clinical Microbiology 47(7): 2351-2352.
Yang, Y.， H. Wang, et al. (2009). "Neuropathology in 2 cases of fatal enterovirus type 71 infection from a recent epidemic in the People's Republic of China: a histopathologic? immuno histo chemic al , and reverse transcription polymerase chain reaction study. " Human Pathology 40(9): 1288-1295.
Zhang, Y.， D. Wang, et al. (2010). "Molecular Evidence of Persistent Epidemic and Evolution of Subgenotype Bl Coxsackievirus A16-Associated Hand? Foot, and Mouth Disease in China." Journal of Clinical Microbiology 48(2): 619-622.
Zhu， Z.， S. Zhu， et al. (2010). "Retrospective sero epidemio lo gy indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008." Virology Journal 7(1): 300. 中国卫生部 （2010) 手足口病诊疗指南 (2010年版).

Claims

权 利 要 求
1. 一种 P2X受体拮抗剂的用途，用于制备抑制正义单链 RNA病毒微小核糖核酸病 毒的组合物。
2. 如权利要求 1所述的用途， 其特征在于， 用于制备预防、 缓解或治疗手足口病 的组合物。
3. 如权利要求 1或 2所述的用途， 其特征在于， 所述的组合物用于： 全身给药或 肠胃外给药； 较佳地， 所述的组合物用于口服、 静脉注射、 肌肉注射或吸入给药。
4. 一种抑制正义单链 RNA病毒微小核糖核酸病毒的方法， 包括： 给予需要抑制病 毒的对象有效量的 P2X受体拮抗剂。
5. 如权利要求 4所述的方法， 其特征在于， 所述的对象是病毒感染的哺乳动物， 包括肠道病毒 71型或柯萨奇病毒感染的人， 猴子或鼠等， 更佳地是手足口病患者。
6. 如权利要求 1所述的用途， 或如权利要求 4所述的方法， 其特征在于， 所述的 P2X受体拮抗齐 [J选自下组： PPADS , iso-PPADS, PPNDS, Suramin, NF023 , TNP-ATP, NF279, NF157, Evans Blue， 和 /或它们的类似物或衍生物， 和 /或它们的药学上可接 受的盐。
7. 如权利要求 1所述的用途， 或如权利要求 4所述的方法， 其特征在于， 所述的 病毒是肠道病毒。
8. 如权利要求 1所述的用途， 或如权利要求 4所述的方法， 其特征在于， 所述的 病毒是肠道病毒 A ( enterovirus A) 。
9. 如权利要求 1所述的用途， 或如权利要求 4所述的方法， 其特征在于， 所述的 病毒是人肠道病毒 71型和柯萨奇病毒。
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