CN107343888A

CN107343888A - A kind of hydroxycholesterol oxycholesterol 25HC and application thereof

Info

Publication number: CN107343888A
Application number: CN201710569986.8A
Authority: CN
Inventors: 王健伟; 雷晓波; 张珍珍; 肖霞
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2017-07-13
Filing date: 2017-07-13
Publication date: 2017-11-14
Anticipated expiration: 2037-07-13
Also published as: CN107343888B

Abstract

It is a discovery of the invention that 25 hydroxycholesterol oxycholesterols can effectively suppress the duplication of enterovirns type 71 and Enterovirus 68 type.The invention provides the purposes of 25 hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester in prevention and/or treatment enterovirus infection associated diseases and/or the medicine of symptom is prepared, additionally provide method of administration, the formulation suitable for the medicine, the other active components and the drug combination of the medicine and other drugs that the medicine can include；In addition, additionally provide the method for the non-treatment purpose of the enterovirus suppressed with 25 hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester in cell.

Description

A kind of hydroxycholesterol oxycholesterol 25HC and application thereof

Technical field

The invention belongs to biological technical field and chemical field, in particular it relates to which 25- hydroxycholesterol oxycholesterols are used to make The purposes of the medicine of standby anti-enterovirus.

Background technology

Enterovirus genus (Enterovirus) ranges micro ribonucleic acid (RNA) Viraceae (Picornaviridae), is The similar single strand plus RNA virus of a kind of biological character, no coating, genome are about 7.2-8.4kb.More than enterovirus exhale It is portal of entry to inhale road, throat and enteron aisle, first in the lymphoid tissue and peyer patches such as local mucous membrane and pharynx, tonsillotome Preliminary propagation, is then released into blood, forms first time viremia virusemia, diffuses to the target tissue with acceptor, after breeding again, draws Play second of viremia virusemia and clinical symptoms.The acceptor of most enteroviruses is widely distributed in tissue and cell, including nerve System, heart, lungs, pancreas, mucous membrane, skin and other systems, therefore, its diseases range is extensive.

Human enterovirus include poliovirus (Poliovirus), Coxsackie virus (Coxsackievirus), Echovirus (ECHO virus) and new enterovirus, at least 72 serotypes.

One long-chain polypeptide of genome encoding of human enterovirus 71 (Enterovirus A71, EV71), can be divided into Structural proteins area P1 and non-structural protein white area P2 and P3.The virus protease 3CD of P3 codings is processed to P1, cut, and produces Structural proteins VP0, VP1 and VP3, VP0 can further be cracked into VP2 and VP4, and assembling is formed these structural proteins jointly in vivo Viral capsid.EV71 viruses are one of main pathogens of hand-foot-and-mouth disease.Hand-foot-and-mouth disease principal pathogenetic crowd is less than 5 years old baby children Youngster, clinical manifestation are heating, are had sore throat, erythema, blister or even ulcer etc. occur in the area skin such as hand, foot, oral cavity and mucous membrane The gastrointestinal symptoms such as Nausea and vomiting, stomachache, diarrhoea generally occur in infringement, severe EV71 infected patients, and with maincenter god It is comprehensive through systematic complication, including acute flaccid brain paralysis, encephalitis, meningitis, BBE, encephalomyelitis, polio sample A variety of neurogenic diseases such as simulator sickness, aseptic meningitis and neurogenic pulmonary edema, empsyxis, respiratory tract infection, cardiovascular damage The complication such as wound, myocarditis, a small number of EV71 infected patients can also intestinal bleeding caused by center of origin nervous system disorder, with from The relevant slight dysfunction of liver of main neurological disorders, inflammatory reaction, and MOF.

EV71 especially constitutes great threat to the health of the mankind to the health of infant.Even to this day, EV71 Mechanism of causing a disease is not entirely clear that effective preventions are still limited.A few days ago, first EV71 inactivated vaccines (two times of the people in the whole world Body cell) the approval production of state food pharmaceuticals administration general bureau is obtained, the vaccine is ground by Chinese Academy of Medical Sciences's Medical Biology Study carefully institute's independent research (referring to Chinese patent CN 101402944B).Clinical test results show that this vaccine causes to EV71 Hand-foot-and-mouth disease protective rate up to 97.3%, the incidence of disease for effectively reducing Chinese children hand-foot-and-mouth disease, especially reduce The sick severe and death, protection children's life and health are significant.However, ensuing problem is Jin Jinyi The control of hand-foot-and-mouth disease whether can be realized by vaccine.By taking the prevention and control history of polio as an example, before 2000, global range The elimination of polio is realized by large-scale vaccine inoculation, but in recent years, polio is still in multiple states Family and area are popular.Therefore, realize the control of enterovirus is also necessary not only for effective vaccine, the auxiliary of antiviral drugs It is vital.At present clinically often using wide spectrum antiviral drugs and with antipyretic and antidote functions Chinese herbal medicine, it is middle into Medicine treats hand-foot-and-mouth disease, still lacks to enterovirus especially EV71 virus more targetedly medicine.

1 precursor polyprotein of genome encoding of the type of human enterovirus 68 (Enterovirus D68, EV68), then Structural protein VP1-VP4 and NS2 Protein A, 2B, 2C and 3A, 3B, 3C, 3D are constantly cut into by oneself protein enzyme hydrolysis. The common sympton of EV68 virus infection has：Have a running nose, sneeze, cough, courbature, asthma, have difficulty in breathing, heating, throat pain Bitterly, there are erythema, blister and/or ulcer, and other serious symptoms etc. in the area skin such as hand, foot and/or oral cavity and/or mucous membrane. Clinical complication has：The infection of the upper respiratory tract, bronchiolitis, bronchitis and pneumonia, hand-foot-and-mouth disease, herpangina, Pleuralgia, aseptic meningitis, flaccid paralysis, sepsis of the newborn and some serious chronic diseases.EV68 viruses and severe Respiratory disease is relevant, can aggravate asthma and pneumonia, triggers high heat syncope and lethal nervous system infection, single cases Cardiorespiratory failure and central nervous system complication are may occur in which after infection, or even is triggered dead.Meanwhile U.S.'s prevention from suffering from the diseases control Center processed reports a kind of the nervous system disease characterized by gray nucleus is abnormal with muscle weakness, some patientss EV68 detections As a result it is positive.

EV68 breaks out the whole world more in recent years, and induces serious respiratory system and central nervous system disease, causes Serious infection symptoms.EV68 virological Features and mechanism of causing a disease are not fully understood at present.Liu et al. has found antiviral agent Thing pleconaril (Pleconaril) can block EV68 to infect in the cell, be likely to become the drug candidate for the treatment of EV68 infection (Liu Y,Sheng J,et al.Structure and inhibition of EV-D68,a virus that causes respiratory illness in children[J].Science,2015,347(6217):71-74).Do not have so far Specificity is directed to EV68 vaccine, can also be applied without granted safe antiviral therapy in clinical treatment, there is an urgent need to Special effective prevention and medicine are researched and developed to contain the harm of virus.

25- hydroxycholesterol oxycholesterols (25-Hydroxycholesterol, 25HC), also cry 25-HYDROXY CHOLESTEROL, 5- cholestene- 3 β, 25- glycol (5-Cholestene-3 β, 25-diol), chemical formula C₂₇H₄₆O₂, molecular weight 402.65, No. CAS is 2140- 46-7, structural formula are：

25- hydroxycholesterol oxycholesterols are a kind of Native Oxide sterol, are widely present in cell, are produced by cholesterol metabolic, To being played an important role in cellular lipid metabolism and process of immune regulation.At present, 25- hydroxycholesterol oxycholesterols are mainly used in synthesizing or made Other standby cholesterol compounds, are a kind of conventional chemical intermediates.Also there are some researches show it can be used for regulation cholesterol Accumulation.In viral biology research field, there are 25- hydroxycholesterol oxycholesterols to prevent some to have by suppressing cell membrane fusion The report of the cell entry host cell of coating, such as reported virus have HCV (Hepatitis C Virus, HCV), human immunodeficiency virus (Human Immunodeficiency Virus, HIV), zika virus (Zika Virus), Nipah virus (Nipah Virus), Ebola virus (Ebola Virus, EBOV) etc. are (for example, see Yongzhi Chen et al.,Interferon-Inducible Cholesterol-25-Hydroxylase Inhibits Hepatitis C Virus Replication via Distinct Mechanisms,SCIENTIFIC REPORTS, 2014,4,7242:1-8；Chunfeng Li et al.,25-Hydroxycholesterol Protects Host against Zika Virus Infection and Its Associated Microcephaly in a Mouse Model,Immunity,2017(46):446-456；And the A of Chinese patent application CN 105362278).But 25- is there is no at present The report of the anti-enterovirus of hydroxycholesterol oxycholesterol.

The content of the invention

Inventor has found that 25- hydroxycholesterol oxycholesterols can effectively suppress the duplication of EV71 viruses, and in the different time points of infection Add, can effectively suppress the duplication of EV71 viruses.Inventor also found that 25- hydroxycholesterol oxycholesterols are to belonging to enterovirus The EV68 viruses of category are also inhibited.The present invention discloses, and 25- hydroxycholesterol oxycholesterols are expected to be used for preparing new anti-enteron aisle disease Cytotoxic drug.

The invention provides 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester to prepare prevention and/or treatment Purposes in enterovirus infection associated diseases and/or the medicine of symptom.

In addition, present invention also offers the method for administration suitable for the medicine, formulation, other work that the medicine can include Property composition and the drug combination of the medicine and other drugs.

Present invention also offers suppressed with 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester in cell in vitro The method of the non-treatment purpose of enterovirus.

Brief description of the drawings

Fig. 1 shows that 25HC can suppress the duplication of EV71 viruses.The 25HC of various concentrations is added in RD cells, 37 DEG C After placing 1h, 1MOI EV71 is added, harvesting and extract cell total rna after 24h, pass through fluorescence quantitative PCR detection EV71 MRNA transcriptional level (Figure 1A), while the expression of immunoblotting (western blot) detection EV71 structural proteins (Figure 1B).

Fig. 2 display infection different time points, which add 25HC, can suppress the duplication of EV71 viruses.In RD cells by EV71 diseases Before poison infection, infection add the 25HC of various concentrations simultaneously or after infection, infect harvesting after 24h and to extract cell total RNA, carry out fluorescence quantitative PCR detection EV71mRNA transcriptional level.

Fig. 3 shows that 25HC can suppress the duplication of EV68 viruses, but to RSV (Respiratory Syncytial Virus, Respiratory Syncytial Virus(RSV), belong to Paramyxoviridae Pneumovirinae Pneumovirus) duplication do not influence.Will be different dense The 25HC of degree is added in RD cells, after 37 DEG C are placed 1h, be separately added into the EV68 (Fig. 3 A) of various concentrations, RSV (Fig. 3 B), EV71 (Fig. 3 C) viruses, harvesting and cell total rna is extracted after 24h, carry out fluorescence quantitative PCR detection this 3 kinds of viral mRNA Transcriptional level.

Sequence table explanation

SEQ ID NO:1 shows the forward primer for detecting EV71 or EV68.

SEQ ID NO:2 show the reverse primer for detecting EV71 or EV68.

SEQ ID NO:3 show the forward primer for detecting GAPDH.

SEQ ID NO:4 show the reverse primer for detecting GAPDH.

SEQ ID NO:5 show the forward primer for detecting RSV.

SEQ ID NO:6 show the reverse primer for detecting RSV.

Embodiment

In the present invention, " pharmaceutically acceptable salt " refers to the salt of the relative nontoxic of 25- hydroxycholesterol oxycholesterols, and it includes The all possible pharmaceutically acceptable salt of 25- hydroxycholesterol oxycholesterols, these salt see, for example, S.M.Berge, et al. “Pharmaceutical Salts,”J.Pharm.Sci.1977,66,1-19。

In the specific embodiment of the present invention, the pharmaceutically acceptable salt includes 25- hydroxycholesterol oxycholesterols Single salt, or any mixture of the salt of any ratio.

In the specific embodiment of the present invention, the pharmaceutically acceptable salt includes but is not limited to：25- hydroxyls The cholesteric alkali metal salt of base, such as sodium salt or sylvite；Alkali salt, such as calcium salt or magnesium salts；Ammonium salt can connect with providing physiology By cation organic base formed salt, such as with aminoglucose, N- methyl-glucamines, dimethyl-aminoglucose, ethyl-aminoglucose, The salt that hexamethylene diamine, monoethanolamine, methyl amimoacetic acid, lysine, trishydroxymethylaminomethane, the propyl alcohol of amino two are formed；Or quaternary ammonium salt, such as with The salt that tetramethyl-ammonium, tetraethyl ammonium are formed.

In the present invention, term " pharmaceutically acceptable ester " refers in people or other animal bodies or extracorporeal hydrolysis generate The all possible pharmaceutically acceptable ester of 25- hydroxycholesterol oxycholesterols.

In the specific embodiment of the present invention, the pharmaceutically acceptable ester includes 25- hydroxycholesterol oxycholesterols Single ester, or any mixture of the ester of any ratio.

In the specific embodiment of the present invention, the pharmaceutically acceptable ester includes but is not limited to：25- hydroxyls Base cholesterine and the carboxylic acid ester that for example formic acid, acetic acid, tertiary butyric acid, palmitic acid, stearic acid, benzoic acid, phenylacetic acid are formed；With sulfonic acid Such as the ester that methanesulfonic acid, ethyl sulfonic acid, ethionic acid, benzene sulfonic acid are formed；With amino acid such as valine, leucine, isoleucine The ester of formation；And with inorganic acid for example phosphoric acid, sulfuric acid, sulfurous acid formed inorganic ester.

In addition, the present invention includes all possible prodrug of 25- hydroxycholesterol oxycholesterols.

In the present invention, term " prodrug " includes the pharmaceutically acceptable derivates of 25- hydroxycholesterol oxycholesterols, and its own can To be that biological activity or biology are inactive, but its in vivo the retention period be converted (such as by being metabolized or hydrolyzing) and be 25- hydroxycholesterol oxycholesterols.One example of prodrug is the ester or phosphate of 25- hydroxycholesterol oxycholesterols.In the present invention, term " prodrug " Also include the covalent bond form of active component and any carrier, this form can discharge active component in vivo.For example, see Testa,B.and Mayer,J.M.,Hydrolysis in drug and prodrug metabolism:chemistry, biochemistry,and enzymology,Wiley-VCH,Zurich,2003；Bundgaard,Design of Prodrugs,Elsevier(1985)；And E.B.Roche, Bioreversible Carriers in Drug Design, Pergamon Press,1987。

In addition, present invention additionally comprises all possible metabolin of 25- hydroxycholesterol oxycholesterols.

It should be understood that those skilled in the art can be during 25- hydroxycholesterol oxycholesterols be synthesized or after synthesis to 25- hydroxyl courages The structure of sterol carries out a variety of activity transformed without influenceing its anti-enterovirus, such as by one on 25- hydroxycholesterol oxycholesterols Or multiple hydrogen atoms identical or different halogen, C₁-C₆- alkyl, C_3-8- cycloalkyl, hydroxyl, ester group, amino, acyl group, sulphonyl The groups such as base, sulfinyl substitute, and are and for example usually used in by 25- hydroxycholesterol oxycholesterols and pharmaceutically conjugated stable to improve compound The material of property, half-life period etc. are mutually conjugated.It is expected that the transformation of all anti-enterovirus activity for not significantly affecting 25- hydroxycholesterol oxycholesterols Fall within the scope of the present invention.

In the present invention, term " halogen " refers to fluorine, chlorine, bromine or iodine.

In the present invention, term " C₁-C₆- alkyl " refers to the straight or branched alkyl with 1-6 carbon atom, including but It is not limited to：Methyl, ethyl, n-propyl, isopropyl, normal-butyl, sec-butyl, the tert-butyl group, amyl group, isopentyl, hexyl, 2- methyl fourths Base, 1- methyl butyls, 1- ethyl propyls, 1,2- dimethyl propyls, neopentyl, 1,1- dimethyl propyls, 4- methyl amyls, 3- first Base amyl group, 2- methyl amyls, 1- methyl amyls, 2- ethyl-butyls, 1- ethyl-butyls, 3,3- dimethylbutyls, 2,2- dimethyl Butyl, 1,1- dimethylbutyls, 2,3- dimethylbutyls, 1,3- dimethylbutyls or 1,2- dimethylbutyls.

In the present invention, term " C_3-8- cycloalkyl " refers to the cycloalkyl with 3-8 carbon atom, including but not limited to ring Propyl group, cyclobutyl, cyclopenta, cyclohexyl and suberyl.

In the specific embodiment of the present invention, the medicine is used for mammal, people or birds.

In a preferred embodiment, the medicine is used for people, ox or pig.

In a further preferred embodiment, the medicine is used for people.

In an especially preferred embodiment, the medicine is used for the children of less than 16 years old, or the medicine is used In the infant of less than 5 years old.

In the present invention, term " enterovirus " refers to the virus for the enterovirus genus for belonging to Picornaviridae.

The present invention a specific embodiment in, the enterovirus include Coxsackie virus A group 16,4,5,7, 9th, 10 type, the type of B groups 2,5,13；Echovirus and Enterovirus 68,71 types.

In a preferred embodiment, the enterovirus is Enterovirus 68 type or enterovirns type 71.

In the specific embodiment of the present invention, the disease includes enterovirns type 71 infection associated diseases, These diseases include but is not limited to hand-foot-and-mouth disease, acute flaccid brain paralysis, encephalitis, meningitis, BBE, encephalomyelitis, ridge Marrow poliomyelitis sample syndrome, aseptic meningitis.

In a preferred embodiment, the disease is hand-foot-and-mouth disease.

In the specific embodiment of the present invention, the symptom includes symptom caused by enterovirns type 71 infection, These symptoms include but is not limited to generate heat, and have sore throat, the area skin such as hand, foot and/or oral cavity and/or mucous membrane occur erythema, Blister and/or ulcer etc. are damaged, and the gastrointestinal symptom such as Nausea and vomiting, stomachache, diarrhoea, and neurogenic pulmonary edema, lung go out Blood, respiratory tract infection, cardiovascular injury, myocarditis, intestinal bleeding, dysfunction of liver and MOF.

In a preferred embodiment, the symptom includes but is not limited to generate heat, and has sore throat, hand, foot and/or mouth There is erythema, blister and/or ulcer in the area skins such as chamber and/or mucous membrane.

In the specific embodiment of the present invention, the disease includes Enterovirus 68 type infection associated diseases, These diseases include but is not limited to the infection of the upper respiratory tract, bronchiolitis, bronchitis, pneumonia, hand-foot-and-mouth disease, herpetic pharynx Laryngitis, pleuralgia, aseptic meningitis, flaccid paralysis, sepsis of the newborn.

In the specific embodiment of the present invention, the symptom includes symptom caused by the infection of Enterovirus 68 type, These symptoms include but is not limited to：Have a running nose, sneeze, cough, courbature, asthma, have difficulty in breathing, heating, have sore throat, There is erythema, blister and/or ulcer in the area skins such as hand, foot and/or oral cavity and/or mucous membrane.

Herein, " prevention " refers to reduce risk or delay subject illness or the delay disease that subject is attacked by a disease The Primary preventive intervention of shape time of occurrence.

Herein, " treatment " refers to the therapeutic intervention of the state of development for the disease or symptom for improving subject, including Make the disease or resolution of symptoms of subject, slow down the process of subject's disease or symptom, mitigate the tight of subject's disease or symptom Weight degree, or reduce the cell, physiology or the biochemistry cause of disease or mechanism for causing disease or symptom.

In the specific embodiment of the present invention, the medicine is included in 25- hydroxycholesterol oxycholesterols or its salt or ester At least one as active component.

In a preferred embodiment, the medicine includes the active component of therapeutically effective amount.

In the present invention, it is desired to refer to that the amount of included active component can be realized effectively for term " therapeutically effective amount " Therapeutic response and the subject to being treated there is no toxicity.For example, " therapeutically effective amount " refer to be enough in subject's body it is pre- The anti-and/or amount for the treatment of enterovirus infection associated diseases and/or illness, and do not cause substantive cell in subject's body Toxic action.Serious journey of the therapeutically effective amount of active component depending on the health of specific subject, disease and/or symptom Interaction of degree and concurrent treatment etc..

In the specific embodiment of the present invention, the medicine also includes other active components.

In a preferred embodiment, the other active components are selected from following one or more：Antivirotic, Antiseptic, anodyne, antipyretic, antiinflammatory, anesthetic etc..

In a preferred embodiment, occur between the active component of the medicine or do not occur knot chemically Close, condense, conjugated or fusion.

In the specific embodiment of the present invention, the medicine can also pharmaceutically connect comprising inert, nontoxic The auxiliary material received.

In the present invention, " pharmaceutically acceptable auxiliary material " refers to relative nontoxic and harmless auxiliary material, any to be drawn by auxiliary material The side effect risen is all without the beneficial effect for weakening active component.By manner known in the art by auxiliary material and active component knot Close so that active component is converted into desired formulation.

In the specific embodiment of the present invention, the medicine is configured to quick-release, sustained release and time controlled released Formulation.

The present invention a specific embodiment in, the medicine by oral administration, parenteral, part, through eye, oral cavity, The approach such as sublingual or rectum are administered.

According to desired method of administration, the medicine can be formulated as suitable agent according to methods known in the art Type, for example, see A.R.Gennaro, Remington:The Science and Practice of Pharmacy, Lippincott Williams&Wilkins,21st Edition,2005；And L.V.Allen, Jr.et al., Ansel ' s Pharmaceutical Dosage Forms and Drug Delivery Systems,8th Edition, Philadelphia,PA:Lippincott,Williams&Wilkins,2004。

In a preferred embodiment, by the oral administration of drugs, solid or liquid preparation are formulated as such as Capsule, pill, tablet, lozenge, lozenge, melt, powder, solution, supensoid agent or emulsion.

In another preferred embodiment, it is subcutaneous, quiet through parenteral route using the medicine as injection type The approach administration such as arteries and veins is interior, intraocular, intrasynovial, intramuscular or intraperitoneal.

In another preferred embodiment, the controlled release preparation for parenteral includes lipid known in the art Body, polymerizing microballoons and polymeric gel preparation.

In another preferred embodiment, the ointment being locally administered, transdermal patch, suction is made in the medicine Agent, nasal spray, suppository etc..

Those skilled in the art can use this area routinely to make it will be appreciated that when preparing medicine of the present invention Auxiliary material is formulated as desired formulation, and such auxiliary material includes but is not limited to：Carrier, excipient, solvent, diluent, Surfactant, thickener, flavor enhancement, preservative, buffer, dispersant, wetting agent, emulsifying agent, suspending agent, disintegrant, increasing Stick, adsorbent, colouring agent, antioxidant, oil, aliphatic acid, fatty acid ester, soaps, detergent, fining agent, encapsulated reagent, Ointment bases, chelating agent, sweetener, retention agent, aerosol propellant, air displacer, jointing material, penetration enhancers, increasing Mould agent, curing agent, tablet antitack agent or polishing agent (for example, see Remington's Pharmaceutical Sciences, E.W.Martin,Mack Publishing,Easton,PA,19^th Edition(1995))。

In the specific embodiment of the present invention, the medicine is included in kit.

In a preferred embodiment, the kit also includes label and/or specification, shows the medicine Therapeutic purposes and/or therapeutic modality.

In the specific embodiment of the present invention, the medicine and other drugs administering drug combinations.

The joint does not cause unacceptable side effect.

In a preferred embodiment, the other drugs, which include other, can prevent and/or treat enterovirus Infect associated diseases and/or the medicine of symptom.

In a preferred embodiment, the other drugs include but is not limited to antiviral drugs, immunomodulator, Anti-infectious agent, analgesic-antipyretic, vaccine, Chinese herbal medicine, Chinese patent drug, probiotics, vitamin, trace element.

In a further preferred embodiment, the other drugs include but is not limited to EV71 inactivated vaccines, EV71 subtracts Malicious vaccine, gamma-globulin, recombinant human interferon alpha 2, RhIL-2, ACV, GCV, Ribavirin, aureomycin Ointment, Moroxydine Hydrochloride, Amoxicillin, paracetamol, montmorillonite powder, Cydiodine buccal tablet, cod-liver oil, Radix Isatidis, ice boron Dissipate, ZHUSHEYONG SHUANGHUANGLIAN, watermelon crystal spray, Qing kailing, Pudilan, vitamin B2, vitamin C.

In a preferred embodiment, the administering drug combinations can be administered simultaneously or be administered in succession.

Drug given alone of the present invention or with dosage required during other drugs administering drug combinations, order of administration and Administration number of times can be determined by those skilled in the art by the therapeutic test of routine.

Present invention also offers suppressed with 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester in cell in vitro The method of the non-treatment purpose of enterovirus, methods described include：In enterovirus with before cells contacting and/or contacting simultaneously And/or after contact, make 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester and cells contacting；Preferably, 25- hydroxyls Base cholesterine or the concentration of its pharmaceutically acceptable salt or ester are 1.5-15 μM；It is highly preferred that 25- hydroxycholesterol oxycholesterols or its medicine The concentration of acceptable salt or ester is 3-12 μM on.

Wherein, it is for example above to 25- hydroxycholesterol oxycholesterols and its regulation of pharmaceutically acceptable salt and ester and enterovirus It is described.

In the specific embodiment of the present invention, the suppression includes the duplication for suppressing enterovirus or enteron aisle disease Cytopathy caused by poison.

In the specific embodiment of the present invention, the cell is the cell of people.

In a preferred embodiment, the cell is human rhabdomyosarcoma's cell.

It should be understood that can be in any combination between each group embodiment as described above.

Below, it is come exemplary description embodiments of the present invention, following examples by embodiment and with reference to accompanying drawing For explaining rather than limiting the invention in any way.If not otherwise specified, reagent used in embodiment is conventional city Sell reagent, detection method used is detection method well known by persons skilled in the art in embodiment.In example below not Detailed Experimental method is indicated, according to normal condition and method, such as Molecular Cloning: A Laboratory room handbook (Sambrook, et al.New York:Cold Spring Harbor Laboratory Press, 1989) side that method or reagent manufacturer provide described in Method.

Embodiment

Influences of the embodiment 1.25HC to EV71 virus replications

RD cells (human rhabdomyosarcoma's cell, purchased from U.S. ATCC (American type culture Collection viral (the Beijing Strains of EV71 are inoculated with after)) being handled through various concentrations 25HC (H1015, purchased from sigma companies of the U.S.) BJ/CHN/2008), by fluorescence quantitative PCR detection EV71 mRNA level in-site, and EV71 viruses are detected by immunoblotting Structural proteins expression.Experiment sets 3 repetitions, and experimental result is averaged.Specific method is as follows：

1.1 virus amplification

Take EV71 30 μ l of virus and 10%FBS (hyclone, purchased from Hyclone companies of the U.S., article No. 30396) DMEM Culture medium (is purchased from Gibco companies of the U.S., article No.：C11965500) combined inoculation is in T75 (growth area 75cm²) blake bottle In 80%-90% abundance (the stretching, extension density of unit area inner cell fraction, i.e. cell in culture plate reaches total culture plate bottom The 80-90% of area) RD cells in, 5%CO at 37 DEG C₂It is incubated, passes through inverted microscope (model daily：7S100, it is purchased from Japanese Nikon company) (cytopathic effect, CPE show as that space between cells becomes big, cell becomes big and become to observation cytopathy Circle).Until after 80%-90% RD cytopathies, sick cell precipitation and supernatant are collected, in -80 DEG C of multigelations 3 times, 12000rpm centrifugations 10min removes cell fragment, will discharge virulent supernatant packing, and be stored in standby in -80 DEG C.

1.2 25HC handle RD cells

0 μM, 1.5 μM, 3 μM, 6 μM or 12 μM of 25HC is separately added into the RD cells of 80%-90% abundance and (uses ethanol Prepare), the 5%CO at 37 DEG C₂Cultivate 1h.

1.3 virus inoculation

1MOI (multiplicity of infection, infection multiplicity) EV71 viruses are taken to be mixed with 10%FBS DMEM It is inoculated in cell.5%CO at 37 DEG C₂Discard culture medium after being incubated 24h, harvesting, for follow-up quantitative fluorescent PCR and Immunoblotting detects.

1.4 quantitative fluorescent PCR

(1) cell total rna extracts：

In each group cell harvested in 1.3,1ml Trizol (total serum IgE extraction agents, purchased from the U.S. are separately added into Invitrogen companies), room temperature places 10min；200 μ l chloroforms are added, room temperature places 3min after acutely vibrating 15s, makes it certainly Right split-phase；12,000g centrifuges 15min at 4 DEG C, and 500 μ l supernatants are carefully transferred into new Eppendorf manages (no RNase (RNAase) in), isometric isopropanol is added, places 10min after mixing on ice；12,000g centrifuges 10min at 4 DEG C, carefully Abandon supernatant；The ethanol of 1ml 75% is added into RNA precipitate and ((is purchased from Amresco companies of the U.S., article No. with the water without RNase E476) prepare), the gentle precipitation that suspends；7,500g centrifuges 5min at 4 DEG C, carefully abandons supernatant, drying at room temperature precipitation；Into precipitation The water that 50 μ l are free of RNase is added, room temperature places 10min, and fully to dissolve RNA, measure concentration is 200-300ng/ μ l (with U.S. StateSpectrophotometric determination, model ND-1000), it is sub-packed in -80 DEG C and saves backup.

(2) reverse transcription reaction (RT)：

Take 2 μ g RNA and 0.5 μ g oligo (dT)₁₈(being purchased from Japanese Takara companies, article No. RR52A) mixes, and is allowed to Cumulative volume is 15 μ l, 5min at 70 DEG C.1min is placed on ice.According to the M-MLV reverse transcriptase specifications of promega companies of the U.S. The reverse transcription system of middle offer carries out reverse transcription reaction：

Above-mentioned two system is mixed, 60min at 42 DEG C.

(3) quantitative fluorescent PCR：

Internal reference is made with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), passes through fluorescence quantitative PCR detection EV71 mRNA level in-site.

Following primer is used to detect EV71：

Forward primer：5'-ACATGGTGTGAAGAGyCTAyTGAGCT-3', wherein y refer to degeneracy base, represent C or T, sequence Row such as SEQ ID NO:Shown in 1；

Reverse primer：5'-ACACGGACACCCAAAGTAGTCGGTTCCGC-3', sequence such as SEQ ID NO:Shown in 2.

Following primer is used to detect GAPDH：

Forward primer：5'-CGGAGTCAACGGATTTGGTCGTA-3', sequence such as SEQ ID NO:Shown in 3；

Reverse primer：5'-AGCCTTCTCCATGGTGGTGAAGAC-3', sequence such as SEQ ID NO:Shown in 4.

According to 2 × SYBR Premix ExTaq (PCR premixed liquids) specification configuration PCR reactions of Japanese Takara companies System：

Use quantitative real time PCR Instrument (BIO-RAD companies of U.S. CFX^TMOptics Module) enter performing PCR amplification program： 95℃3min；95 DEG C of 5s, 55 DEG C of 15s, 60 DEG C of 30s+ read plates, totally 45 circulations；95 DEG C, 10s；65 DEG C -95 DEG C of melting curve, 5s It is incremented by+read plate.

(4) data processing

Amplification procedure and fluorescence signal detection, the storage of data and analysis by the PCR instrument and carry software completion.Often Individual sample amounts PCR Ct values subtract respective sample GAPDH Ct, and the calculating of the multiple change of sample amplification uses 2^{^ΔCt}Method.

As a result as shown in Figure 1A.As a result show, 25HC can effectively suppress the duplication of EV71 viruses, with the increasing of 25HC concentration Add, suppress the effect increase of EV71 viruses, when 25HC concentration reaches 3 μM, the duplication of EV71 viruses has almost been totally constrained.

1.5 immunoblotting

Internal reference is made with beta-actin (β-actin), carries out immunoblotting detection：

(1) each group cell harvested in 1.3 is handled as follows respectively：Every 1.5 × 10⁶Individual cell adds 100 μ l 1 × passive lysate (passive lysis buffer, by being prepared purchased from 5 × passive lysate of Promega companies of the U.S.：1 The 5 of times volume × passive lysate adds the water of 4 times of volumes, is used after mixing), gently mix, lysis at room temperature 30min；At 4 DEG C 12000g centrifuges 10min, collects supernatant, and (the BCA eggs purchased from Thermo companies of the U.S. are used with BCA protein quantifications method is quantitative White quantification kit (23225), specific method referring to kit specification) cell pyrolysis liquid protein concentration, by each sample The protein concentration of product is transferred to consistent (2 μ g/ μ l), add 10 μ l 6 × sample-loading buffer (300mM Tris-HCl (pH 6.8), 600mM dithiothreitol (DTT)s, 12% (W/V) SDS, 0.6% (W/V) bromophenol blue, 60% (V/V) glycerine) protein sample is prepared into, - 20 DEG C of preservations are placed in after 100 DEG C of heat denatured 5min；

(2) protein sample is separated with 12% polyacrylamide gel electrophoresis (SDS-PAGE), then will through 400mA constant currents Protein transfer is on nitrocellulose (NC) film；

(3) after the completion of transferring film, NC films is closed into 2h through 5% skim milk/PBST (PBS 0.1%Tween20) room temperature, added Primary antibody dilution (presses 1 with 5% skim milk:1000 dilutions) 4 DEG C overnight, then washed three times with 1 ‰ Tween PBST, every time 5min, adding secondary antibody dilution, (5% skim milk presses 1:10000 dilutions), room temperature lucifuge is incubated 30min；1‰Tween PBST Wash three times, each 10min；Swept afterwards with Odyssey (the Dual band IR laser imaging system of Li-COR companies of the U.S.) Retouch identification.Wherein, for EV71 structural proteins and beta-actin, the primary antibody used respectively is the anti-EV71 structural proteins of mouse (MAB979, purchased from Millipore companies of the U.S.) and mouse anti-beta-actin monoclonal antibodies (A5441, purchased from the U.S. Sigma companies), the secondary antibody used respectively is that anti-mouse IgG Dy800 secondary antibodies (are purchased from Li-COR companies of the U.S., article No.：926- 32212) and anti-mouse IgG Dy680 secondary antibodies (are purchased from Li-COR companies of the U.S., article No.：926-68070).

As a result as shown in Figure 1B.As a result show, with the increase of 25HC concentration, EV71 structural proteins VP0 and VP2 amount by It is decrescence few.

Embodiment 2. infects different time points and adds influences of the 25HC to EV71 virus replications

RD cells by before EV71 virus infection, infection simultaneously, after infection, the 25HC of various concentrations is separately added into, by glimmering Fluorescent Quantitative PCR detects EV71 mRNA level in-site.Experiment sets 3 repetitions, and experimental result is averaged.Specific method is as follows：

The 1h after 1h (before infecting), inoculation (infecting simultaneously), inoculation simultaneously before 1MOI EV71 virus inoculations (after infecting), adds 6.25 μM or 12.5 μM of 25HC in RD cells respectively, and each group experiment is all provided with a virus inoculation and is not added with 25HC control.Virus inoculation method is the same as embodiment 1.3.The each group 5%CO at 37 DEG C after infection₂24h is incubated, discards culture Base, harvesting.To each group cell of harvest, by fluorescence quantitative PCR detection EV71 mRNA level in-site, detection method is the same as implementation Example 1.4.

As a result it is as shown in Figure 2.As a result show, before RD cell infection viruses, infection simultaneously, infection after, 25HC is equal The duplication of EV71 viruses can effectively be suppressed.Cell is handled with 6.25 μM or 12.5 μM of 25HC before infection, EV71 viruses Duplication is almost totally constrained；Cell is handled with 12.5 μM of 25HC simultaneously in infection, the duplication of EV71 viruses is almost complete Suppress.

Influences of the embodiment 3.25HC to EV68 virus replications

RD cells are inoculated with EV68 viruses (No. GeneBank respectively after various concentrations 25HC processing：KF726085.1)、RSV Viral (RSV-A2 strain virus, purchased from U.S. ATCC) or EV71 viruses, pass through the mRNA water of fluorescence quantitative PCR detection different virus It is flat.Experiment sets 3 repetitions, and experimental result is averaged.Specific method is as follows：

3.1 virus amplification

The amplification method of EV68 viruses is identical with EV71's, as described in embodiment 1.1.The amplification method of RSV viruses is as follows：

HEp-2 cells (human laryngeal cancer cell, purchased from ATCC, numbering：ATCC CCL-23, cell concentration 1.8 × 10⁵Individual/ml) It is inoculated in 75cm²Tissue Culture Flask.CO at 37 DEG C₂Culture to cell confluency rate reaches 60-70% in incubator.Discard former culture Base, 3ml Opti-MEM (Gibco Products, article No.：31985-070) wash 1 time.Add 200-250 μ l RSV strains and 2ml Opti-MEM, mix.CO at 37 DEG C₂2.5h is incubated in incubator, rocking cell bottle per 15min makes liquid distribution uniform.It is changed to Maintaining liquid (the Opti-MEM culture mediums containing 2% hyclone and 1% mycillin), CO at 37 DEG C₂48- is cultivated in incubator 72h.To cytopathy during obvious and about 30% cells float, be placed in it is quick-frozen in ethanol-dry ice mixture, to cell bottle in liquid Fully charge.37 DEG C of water-baths are melted rapidly, are again placed in ethanol-dry ice mixture to liquid fully charge in cell bottle.37 DEG C water-bath is melted rapidly, 4 DEG C of centrifugation 10min of 3000rpm after blowing and beating repeatedly.Supernatant is taken, is dispensed, -80 DEG C of preservations.

3.2 25HC handle RD cells

0 μM, 3 μM or 12 μM of 25HC, the 5%CO at 37 DEG C are separately added into the RD cells of 80%-90% abundance₂Training Support 1h.

3.3 virus inoculation

2MOI EV68 viruses or 2MOI RSV viruses or 1MOI EV71 viruses are inoculated with into RD cells respectively.3 kinds The inoculation method of virus is identical, as described in embodiment 1.3.5%CO at 37 DEG C₂Culture medium, harvesting are discarded after being incubated 24h.

3.4 quantitative fluorescent PCR

To each group cell of harvest, pass through the mRNA of fluorescence quantitative PCR detection EV68 viruses, RSV viruses and EV71 viruses Level, detection method is as described in embodiment 1.4.Primer for detecting EV68 is identical with the primer for detecting EV71, sequence Such as SEQ ID NO:1 and SEQ ID NO:Shown in 2；Primer for detecting RSV is as follows：

Forward primer：5'-AGATCAACTTCTGTCATCCAGCAA-3', sequence such as SEQ ID NO:Shown in 5；

Reverse primer：5'-TTCTGCACATCATAATTAGGAGTRTCAAT-3', sequence such as SEQ ID NO:Shown in 6.

3 kinds of viral testing results are respectively as shown in Fig. 3 A, 3B, 3C.As a result show, 25HC also can effectively suppress to belong to together In the duplication of the EV68 viruses of enterovirus genus, with the increase of 25HC concentration, suppress the effect increase of EV68 viruses；And for not Belong to the RSV viruses of enterovirus genus, 25HC does not have inhibitory activity to it.

Above example is exemplary embodiment, be should not be construed as limiting the invention.Those skilled in the art can Recognize, any modification, equivalent substitution and improvements made within the spirit and principles in the present invention etc., be all contained in the present invention Within the scope of.In addition, unless otherwise defined, whole technologies used herein and scientific terminology be respectively provided with the present invention belonging to The identical meaning that those of ordinary skill in field is generally understood.If there is contradiction, determine what is included with this specification Justice is defined.All publication, patent application, patent and the full content of document and all publication that they are quoted herein Thing, patent application, patent and document are included herein by reference.

Sequence table

<110>Institute of Pathogen Biology, Chinese Academy of Medical Sciences

<120>A kind of hydroxycholesterol oxycholesterol 25HC and application thereof

<130> CP1170200/CB

<160> 6

<170>PatentIn version 3s .3

<210> 1

<211> 26

<212> DNA

<213>Artificial sequence

<400> 1

acatggtgtg aagagyctay tgagct 26

<210> 2

<211> 29

<212> DNA

<213>Artificial sequence

<400> 2

acacggacac ccaaagtagt cggttccgc 29

<210> 3

<211> 23

<212> DNA

<213>Artificial sequence

<400> 3

cggagtcaac ggatttggtc gta 23

<210> 4

<211> 24

<212> DNA

<213>Artificial sequence

<400> 4

agccttctcc atggtggtga agac 24

<210> 5

<211> 24

<212> DNA

<213>Artificial sequence

<400> 5

agatcaactt ctgtcatcca gcaa 24

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence

<400> 6

ttctgcacat cataattagg agtrtcaat 29

Claims

1.25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester are preparing prevention and/or treatment enterovirus infection institute Cause the purposes in the medicine of disease and/or symptom.

2. the purposes of claim 1, wherein the medicine is used for people；Preferably, the medicine is used for the infant of less than 5 years old.

3. any one of claim 1-2 purposes, wherein the enterovirus is Enterovirus 68 type or enterovirns type 71.

4. any one of claim 1-3 purposes, wherein the disease include hand-foot-and-mouth disease, acute flaccid brain paralysis, encephalitis, Meningitis, BBE, encephalomyelitis, poliomyelitis-like syndrome, aseptic meningitis, the infection of the upper respiratory tract, capillary branch Tracheitis, bronchitis, pneumonia, herpangina, pleuralgia, flaccid paralysis, sepsis of the newborn；Preferably, it is described Disease is hand-foot-and-mouth disease.

5. any one of claim 1-4 purposes, wherein the symptom includes heating, have sore throat, hand, foot and/or oral cavity There are the digestive systems such as the infringement such as erythema, blister and/or ulcer, Nausea and vomiting, stomachache, diarrhoea Deng area skin and/or mucous membrane Symptom, and neurogenic pulmonary edema, empsyxis, respiratory tract infection, cardiovascular injury, myocarditis, intestinal bleeding, liver function are different Normal and MOF, have a running nose, sneeze, cough, courbature, asthma, expiratory dyspnea；Preferably, the symptom Including heating, have sore throat, erythema, blister and/or ulcer occur in the area skin such as hand, foot and/or oral cavity and/or mucous membrane.

6. any one of claim 1-5 purposes, wherein the medicine and other drugs administering drug combinations；Preferably, it is described its His medicine, which includes other, can prevent and/or treat enterovirus infection associated diseases or the medicine of symptom；Or it is described other Medicine includes：Antiviral drugs, immunomodulator, anti-infectious agent, analgesic-antipyretic, vaccine, Chinese herbal medicine, Chinese patent drug, probiotics, Vitamin, trace element；Preferably, the other drugs include：EV71 inactivated vaccines, EV71 attenuated vaccines, gamma-globulin, again Group human interferon, RhIL-2, ACV, GCV, Ribavirin, chlorotetracycline ointment, Moroxydine Hydrochloride, Ah Amdinocillin, paracetamol, montmorillonite powder, Cydiodine buccal tablet, cod-liver oil, Radix Isatidis, Bingpeng San, ZHUSHEYONG SHUANGHUANGLIAN, west Melon frost spray, Qing kailing, Pudilan, vitamin B2, vitamin C.

7. suppress the non-treatment of the enterovirus in cell in vitro with 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester The method of purpose, methods described include：Enterovirus with before cells contacting and/or contact while and/or contact after, make 25- hydroxycholesterol oxycholesterols or its pharmaceutically acceptable salt or ester and cells contacting.

8. the method for claim 7, wherein 25- hydroxycholesterol oxycholesterols or the concentration of its pharmaceutically acceptable salt or ester are 1.5-15 μM；Preferably, 25- hydroxycholesterol oxycholesterols or the concentration of its pharmaceutically acceptable salt or ester are 3-12 μM.

9. any one of claim 7-8 method, wherein the cell is the cell of people；Preferably, the cell is people's horizontal stroke Line muscle oncocyte.

10. any one of claim 7-9 method, wherein the enterovirus is Enterovirus 68 type or Enterovirus 71 Type.