CN107789344A - A kind of inhibitor of Ebola virus - Google Patents
A kind of inhibitor of Ebola virus Download PDFInfo
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- CN107789344A CN107789344A CN201711012931.3A CN201711012931A CN107789344A CN 107789344 A CN107789344 A CN 107789344A CN 201711012931 A CN201711012931 A CN 201711012931A CN 107789344 A CN107789344 A CN 107789344A
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- ebola
- ebola virus
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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
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
Abstract
The invention belongs to pharmaceutical technology field.Especially, the purposes for preventing and/or treating Ebola virus infection or the disease (for example, Ebola hemorrhagic fever) caused by Ebola virus infects is used for the present invention relates to the bicyclic aminated compounds with formula (I) structure.The application further relates to that there is the bicyclic aminated compounds of formula (I) structure to be used for the purposes for preparing prevention and/or the pharmaceutical composition of the infection for the treatment of Ebola virus or the disease (for example, Ebola hemorrhagic fever) caused by Ebola virus infects.
Description
Technical field
The invention belongs to pharmaceutical technology field.Especially, the present invention relates to the bicyclic aminated compounds with formula (I) structure
For prevent and/or treat Ebola virus infection or by Ebola virus infect caused by disease (for example, Ebola's bleeding
Heat) purposes.The bicyclic aminated compounds that the application further relates to have formula (I) structure is used for preparation prevention and/or treatment angstrom is rich
Draw the use of the pharmaceutical composition of virus infection or the disease (for example, Ebola hemorrhagic fever) caused by Ebola virus infects
On the way.
Background technology
Ebola hemorrhagic fever (EBHF) is viral hemorrhagic fever most fatal in the world today, and the infected's symptom is with being all fibre
It is very much like to tie up the Marburg virus of Viraceae, including the change of nausea,vomiting,diarrhea, the colour of skin, Muscular stiffness, internal bleeding, body
External hemorrhage, fever etc., fatal rate is up to 50-90%, because of its high fatal rate, difficult curative and depositing without effective medicine and vaccine
In the extensive concern by medical field and attention all the time.
Cause Ebola hemorrhagic fever for Ebola virus, belonging to has coating, un-segmented property sub-thread minus-stranded rna virus.Most
Non- remote western Ebola river area in was broken out earlier than 1976, is hence obtained one's name.Virus can be by straight with patient body fluid
Contact, or contact and infect with patient skin, mucous membrane etc..Virus latency generally only had 5 to 10 days up to 2 to 21 days.
1994-1996,2001-2005,2007-2008 Ebola virus occur discontinuity prevalence, 2014 years Ebola viruses in Africa
Spread in West Africa, and sounded the alarm in the whole world.
Active drug and the vaccine listing of anti-Ebola virus are there is no at present.Especially, due to the biology of Ebola virus
Safe class for 4 grades, it is necessary to carry out correlative study to it in 4 grades of laboratories, deposit by so personal safety not only to researcher
In grave danger, the research and development of active drug are also greatly limit.Therefore, the clinical common medicine for finding anti-Ebola virus is just compeled
The eyebrows and eyelashes.
The content of the invention
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The implication that personnel are generally understood that.Also, the operating procedures such as cell culture used herein, biochemistry, cell biology
It is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, relational language is provided below
Definition and explanation.
As used herein, term " Ebola virus (Ebola virus, EBOV) " refers to, category filamentous virus section
(Filoviridae) a kind of minus-stranded rna virus, it is to cause Ebola hemorrhagic fever (EBHF, also known as ebola disease viral disease
(EVD) pathogen).
As used herein, term " pharmaceutically acceptable salt " refers to, is deposited in compound provided by the present invention
Basic functionality (such as tertiary amine group) and the salt of appropriate inorganic or organic anion (acid) formation, and including but
It is not limited to, inorganic acid salt, such as hydrofluoride, hydrochloride, hydrobromate, hydriodate, nitrate, perchlorate, sulfate, phosphorus
Hydrochlorate etc.；Rudimentary alkyl sulfonate, such as mesylate, fluoroform sulphonate, esilate；Arylsulphonate, as benzene sulfonate,
P-TOLUENE SULFO ACID 99's salt etc.；Acylate, as acetate, malate, fumarate, succinate, citrate, tartrate,
Oxalates, maleate etc.；Amino-acid salt, such as glycinate, trimethylglycine salt, arginine salt, ornithine salt, glutamic acid
Salt, aspartate etc..
As used herein, term " effective dose " refers to, it is sufficient to obtains or at least partly obtains intended effect
Amount.For example, " prevention disease effective dose " refers to, it is sufficient to prevent, prevent or postpone disease (for example, Ebola virus infection or by
Ebola virus infection caused by disease) generation amount；" treatment condition effective amount " refers to, it is sufficient to healing or at least part
Prevent the disease of the patient with disease (for example, Ebola virus infection or disease caused by Ebola virus infects)
With the amount of its complication.Effective dose as measure is completely within the limit of power of those skilled in the art.For example, for controlling
Purposes is treated effectively to measure depending on the severity of disease to be treated, the overall status of the immune system of patient oneself, patient
Ordinary circumstance such as age, body weight and sex, the method for application of medicine, and other treatment being administered simultaneously etc..
As used herein, term " subject " includes but is not limited to various animals, such as mammal, such as ox
Section animal, equine species, caprid, porcine animals, canid, cats, rabbit section animal, rodent (for example,
Mouse or rat), non-human primate (for example, macaque or machin) or people.In some embodiments, the subject
(such as people) infects Ebola virus, or, there is the risk for infecting Ebola virus.
Present inventor has found that the bicyclic aminated compounds with specific structure can significantly inhibit ebola disease first
The infection of poison, there is obvious anti-ebola disease cytotoxic activity, available for preventing and/or treat Ebola virus infection or by angstrom rich
Draw the disease (for example, Ebola hemorrhagic fever) caused by virus infection.And before the application, the anti-Ebola of this kind of compound
Virus activity did not appeared in the newspapers.
Therefore, in one aspect, the invention provides the compound shown in logical formula (I) or its pharmaceutically acceptable salt to use
In in subject prevent and/or treat Ebola virus infection or by Ebola virus infect caused by disease purposes,
Or for preparing the prevention in subject and/or treating Ebola virus infection or the disease caused by Ebola virus infects
The purposes of the pharmaceutical composition of disease；
Wherein, m, x, y are each independently 0,1,2,3 or 4.
In certain preferred aspects, x is identical with y.
In certain preferred aspects, m, x, y are mutually the same.
In certain preferred aspects, x is identical with y, and is 0,1,2 or 3.
In certain preferred aspects, m, x, y are mutually the same, and are 0,1,2 or 3.
In certain preferred aspects, the compound has chemical formula as follows：
In certain preferred aspects, the disease caused by Ebola virus infects is Ebola's bleeding
Heat.
In certain preferred aspects, it is described that Zaire's type (Zaire), the Sudan's type are selected from by Ebola virus
(Sudan), Ben Dibujiao types (Bundibugyo), Ta Yisen crop types () and Christopher Eccleston type (Reston) Forest.
In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt or acylate.At certain
In a little preferred embodiments, the pharmaceutically acceptable salt is inorganic acid salt, such as hydrochloride, sulfate, phosphate, first
Sulfonate or benzene sulfonate.
In certain preferred aspects, described pharmaceutical composition include prevention or therapeutically effective amount it is as described above
Compound.
In certain preferred aspects, described pharmaceutical composition can be any form known to medical domain.Example
Such as, described pharmaceutical composition can be tablet, pill, supensoid agent, emulsion, solution, gel, capsule, pulvis, powder,
Granula, elixir, lozenge, suppository, injection (including parenteral solution, injection sterile powder and concentrated solution for injection), inhalant, spray
The forms such as mist agent.In certain preferred aspects, described pharmaceutical composition be tablet, pill, capsule, granule, dissipate
Agent, solution, supensoid agent, parenteral solution or injection sterile powder.
In certain preferred aspects, described pharmaceutical composition optionally also includes can be used for treating Ebola virus
Infection and/or Ebola hemorrhagic fever other forms of pharmacologically active agents, such as sertraline hydrochloride, clomiphene (Clomiphene),
Toremifene (Toremifene), Favipiravir or NB-DNJ (Miglustat).
Vaccine or medicine known in the art, ratifying currently without any one through FDA available for Ebola virus.Cause
This, in the present invention, statement " can be used for the other pharmaceutical active of the infection for the treatment of Ebola virus and/or Ebola hemorrhagic fever
Agent ", which includes those, to be had potential anti-ebola disease cytotoxic activity and/or is envisaged for Ebola virus in clinical experimental stage
Vaccine or medicine, the example include but is not limited to sertraline hydrochloride, clomiphene (Clomiphene), Toremifene
(Toremifene), Favipiravir, NB-DNJ (Miglustat), and it is described in detail in " Trad MA, et al.J Clin
Virol.2017 Jan；86:Those in 5-13 ".
In certain preferred aspects, described pharmaceutical composition includes the compound as described above of unit dose,
Such as the compound of any amount comprising 1-2000mg, such as 10-1500mg, such as 20-1000mg, such as 50-
500mg, such as 50-100mg.In the present invention, the dosage of the compound can be light according to the state of an illness of patient or subject
The factors such as weight, age, body weight, sex, administering mode and the course for the treatment of are adjusted.
In the present invention, described pharmaceutical composition can be applied by any suitable method known in the art, example
It is such as oral, parenteral, rectum, transpulmonary or administer locally to mode.When for it is oral when, mouth can be made in described pharmaceutical composition
Formulation, such as oral solid formulation, such as tablet, capsule, pill, granule；Or, oral liquid, it is such as oral molten
Liquor, oral suspensions, syrup etc..When oral formulations are made, described pharmaceutical composition can also include suitable filling
Agent, adhesive, disintegrant, lubricant etc.；Especially, in order to improve the wettability of hydrophobic compound and improve its dissolubility,
Described pharmaceutical composition preferably includes hydrophilic polymer, such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose
Or carboxymethyl cellulose (CMC) etc. (HPMC).When for parenteral, injection can be made in described pharmaceutical composition, including
Parenteral solution, injection sterile powder and concentrated solution for injection.When injection is made, described pharmaceutical composition can use existing
Conventional method in pharmaceutical field is produced.When preparing injection, described pharmaceutical composition, which can contain, pharmaceutically may be used
The carrier of receiving such as sterilized water, ringer's solution and isotonic sodium chlorrde solution, also suitably attached can be added according to the property of medicine
Add agent such as antioxidant, buffer and bacteriostatic agent.When for rectally, suppository can be made in described pharmaceutical composition
Deng.During for transpulmonary administration, inhalant or spray etc. can be made in described pharmaceutical composition.
In certain preferred aspects, the subject is mammal, such as non-human primate, such as
People.
In another aspect, the invention provides one kind prevention and/or the infection for the treatment of Ebola virus or by ebola disease
The method of disease caused by poison infection, it includes applying subject in need the change shown in the logical formula (I) of effective dose
Compound or its pharmaceutically acceptable salt；
Wherein, m, x, y are each independently 0,1,2,3 or 4.
In certain preferred aspects, x is identical with y.
In certain preferred aspects, m, x, y are mutually the same.
In certain preferred aspects, x is identical with y, and is 0,1,2 or 3.
In certain preferred aspects, m, x, y are mutually the same, and are 0,1,2 or 3.
In certain preferred aspects, the compound has structure shown below formula.
In certain preferred aspects, the disease caused by Ebola virus infects is Ebola's bleeding
Heat.
In certain preferred aspects, it is described that Zaire's type (Zaire), the Sudan's type are selected from by Ebola virus
(Sudan), Ben Dibujiao types (Bundibugyo), Ta Yisen crop types () and Christopher Eccleston type (Reston) Forest.
In certain preferred aspects, the pharmaceutically acceptable salt is inorganic acid salt or acylate.At certain
In a little preferred embodiments, the pharmaceutically acceptable salt is inorganic acid salt, such as hydrochloride, sulfate phosphates, first
Sulfonate or benzene sulfonate.
In certain preferred aspects, can using compound as described above as pharmaceutical composition carry out prepare and
Using.In certain preferred aspects, described pharmaceutical composition can include prevention or therapeutically effective amount it is as described above
Compound.In certain preferred aspects, described pharmaceutical composition can be any form known to medical domain.Example
Such as, described pharmaceutical composition can be tablet, pill, supensoid agent, emulsion, solution, gel, capsule, pulvis, granule,
Elixir, lozenge, suppository, injection (including parenteral solution, injection sterile powder and concentrated solution for injection), inhalant, spray
Etc. form.In certain preferred aspects, described pharmaceutical composition be tablet, it is pill, capsule, granule, powder, molten
Liquid, supensoid agent, parenteral solution or injection sterile powder.
In the present invention, described pharmaceutical composition can be applied by any suitable method known in the art, example
It is such as oral, parenteral, rectum, transpulmonary or administer locally to mode.When for it is oral when, mouth can be made in described pharmaceutical composition
Formulation, such as oral solid formulation, such as tablet, capsule, pill, granule；Or, oral liquid, it is such as oral molten
Liquor, oral suspensions, syrup etc..When oral formulations are made, described pharmaceutical composition can also include suitable filling
Agent, adhesive, disintegrant, lubricant etc.；Especially, in order to improve the wettability of hydrophobic compound and improve its dissolubility,
Described pharmaceutical composition preferably includes hydrophilic polymer, such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose
Or carboxymethyl cellulose (CMC) etc. (HPMC).When for parenteral, injection can be made in described pharmaceutical composition, including
Parenteral solution, injection sterile powder and concentrated solution for injection.When injection is made, described pharmaceutical composition can use existing
Conventional method in pharmaceutical field is produced.When preparing injection, described pharmaceutical composition, which can contain, pharmaceutically may be used
The carrier of receiving such as sterilized water, ringer's solution and isotonic sodium chlorrde solution, also suitably attached can be added according to the property of medicine
Add agent such as antioxidant, buffer and bacteriostatic agent.When for rectally, suppository can be made in described pharmaceutical composition
Deng.During for transpulmonary administration, inhalant or spray etc. can be made in described pharmaceutical composition.
In certain preferred aspects, methods described optionally also includes applying can be used for treatment Ebola virus sense
The other forms of pharmacologically active agents of dye and/or Ebola hemorrhagic fever, such as sertraline hydrochloride, clomiphene (Clomiphene), support
Rui meter Fen (Toremifene), Favipiravir or NB-DNJ (Miglustat).This other forms of pharmacologically active agents can applied
With being applied prior to, concurrently with, or after compound as described above.
In certain preferred aspects, as above institute can be applied with any amount of 1~2000mg/kg subject's body weight
The compound stated, such as with 1~1500mg/kg body weight/days, 1~1000mg/kg body weight/days, 5~1000mg/kg body weight/days,
5~500mg/kg body weight/days, 5~200mg/kg body weight/days, 5~100mg/kg body weight/days or 10~100mg/kg body weight/days
Amount apply compound as described above.In certain preferred aspects, can with 4 times a day, 3 times a day, daily 2
It is secondary, one time a day, every 1 time on the two or mode 1 times a week apply compound as described above, optionally take the circumstances into consideration weekly or monthly
Repeat dosage regimen as described above.In the present invention, the dosage of the compound can be according to the disease of patient or subject
The factors such as feelings weight, age, body weight, sex, administering mode and the course for the treatment of are adjusted.
In certain preferred aspects, the subject is mammal, such as non-human primate, such as
People.
The beneficial effect of invention
At present due to lacking to Ebola virus infection or the effective medicine of Ebola hemorrhagic fever and treatment means, mankind's sense
The death rate after Ebola virus is contaminated up to 90%.Present inventor is found that tool first by largely studying and screening
There is the compound of anti-ebola disease cytotoxic activity.The bicyclic aminated compounds can significantly interfere with infection of the Ebola virus to cell
Process, and there is significant protective effect to Ebola virus animal model, there is obvious anti-ebola disease cytotoxic activity.Cause
And bicyclic aminated compounds can be used for preventing and/or treat Ebola virus infection or caused by Ebola virus infects
Disease (for example, Ebola hemorrhagic fever), preventing and treating of this discovery for Ebola virus and/or Ebola hemorrhagic fever have great
Meaning.
Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the art
Member will be understood that drawings below and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.With reference to the accompanying drawings
With the following detailed description of preferred embodiment, various purposes of the invention and favourable aspect are to those skilled in the art
It will be apparent.
Brief description of the drawings
Fig. 1 shown in embodiment 2, analysis curves of the EBOV-10 to Ebola's pseudovirus inhibiting rate.Wherein, horizontal seat
Mark represents EBOV-10 activity (μM), and ordinate represents inhibiting rate (%).As a result show, EBOV-10 can be significantly inhibited angstrom
It is rich to draw pseudovirus.
Fig. 2 shown in embodiment 3, the analysis curve of EBOV-10 cytotoxicity.Wherein, abscissa represents EBOV-10
Activity (μM), ordinate represent inhibiting rate (%).As a result show, EBOV-10 is without obvious cytotoxicity.
Fig. 3 shown in embodiment 4, analysis curves of the EBOV-10 to Ebola's live virus inhibiting rate.Wherein, horizontal seat
Mark represents EBOV-10 activity (μM), and ordinate represents inhibiting rate (%).As a result show, EBOV-10 can substantially suppress angstrom
The rich infection for drawing live virus to cell.
Fig. 4 shows in embodiment 5 that antiviral activities of the EBOV-10 in Ebola virus infects In vivo model divides
Analyse result.As a result show, EBOV-10 can substantially suppress infection of the Ebola virus to mouse.
Fig. 5 is shown in embodiment 6, by surface plasma resonance technology to EBOV-10 and Ebola virus memebrane protein
The testing result of binding activity.As a result show, EBOV-10 has certain binding activity to Ebola virus memebrane protein.
Embodiment
The present invention is described referring now to the following embodiment for being intended to illustrate (and non-limiting present invention) of the invention.
Unless specifically stated otherwise, otherwise basically according to known in the art and normal described in various bibliography
Rule method carries out the experiment and method described in embodiment.Unreceipted actual conditions person in embodiment, according to normal condition or system
The condition for making business's suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the routine of acquisition purchased in market
Product.Those skilled in the art know that embodiment describes the present invention by way of example, and are not intended to limit the required guarantor of the present invention
The scope of shield.Entire disclosure case mentioned in this article and other references are merged into herein with its full text by quoting.
The structure of the pseudovirus of embodiment 1.
The 1.1 antiviral drugs screening principles based on pseudovirus
Envelope protein (for example, envelope protein of Ebola virus) in virus be responsible for the acceptors of identification target cells from
And start absorption and the process penetrated, so as to invade cell.In addition, occurred when two kinds of viruses infect a kind of cell simultaneously
Phenotype mixing phenomena prompts the particle surface that a kind of viral cyst membrane can be incorporated into another different virus.Two based on more than
Point, a kind of new technology is generated in the process of research Virus entry cell and its tissue tropism and acceptor etc. --- cape horn fever
Malicious technology.
Current pseudovirus then refers to that a kind of retroviruse can integrate the cyst membrane sugar of another variety classes virus
Albumen, so as to the cyst membrane with exogenous virus formed, and genome remains retroviruse genomic characterization itself
Virus.Pseudovirion is that the virus protein provided by incasing cells carries the viral vector recombinant for being wrapped in foreign gene,
There was only the ability of single infection to target cell, be replication defect type, new virion can not be produced after infection.Though this virus
Cell can be so invaded, but due to being replication defect type after cell is invaded, and the structural proteins for being assembled into virus are outer
What source provided, it is impossible to the self-replacation of virus is carried out, so very safe.Due to pseudovirus biting property of parent and course of infection with
Euvirus is identical, therefore the early process that can be infected with simulated virus, and reporter gene (fluorescein is carried in pseudovirus
Enzyme), various detections and analysis can be fast, easily carried out, the pseudovirus of this laboratory structure can not only be used for studying disease
The relation of poison and host cell, the acceptor of clonal virus, it is often more important that can be used for screening antiviral drugs, evaluation vaccine is exempted from
Effect of epidemic disease etc..
The present invention is using the plasmid and skeleton plasmid cotransfection 293T cells for expressing Ebola virus glycoproteins, so as to obtain
Ebola's pseudovirus.After medicine effect a period of time after this pseudovirus and doubling dilution, pseudovirus sensitive cells is added,
37 DEG C of 5%CO2After continuing culture in incubator 48 hours, luminous value is detected.If medicine prevents viruses adsorption, into thin
Born of the same parents, then sample well luminous value can decay or disappear；If medicine is sent out without antiviral activity, sample well luminous value with virus control
Light value is suitable.
The preparation of 1.2 pseudovirus
A) preparation of Ebola virus
Using transfection reagent Lipofectamine 3000, by envelope plasmid pCDNA3.1-EBOV-ZGP-8A (referring to example
Such as Liu Q, et al.Sci Rep.2017 Mar 30；7:45552.) and skeleton plasmid pSG3 Δs env.cmvFluc (referring to,
Such as Chinese patent application 201510293955.5) according to mass ratio 1：(ATCC is numbered 2 ratio cotransfection 293T cells：
CRL3216), 4~6h or so changes culture medium after transfection, is subsequently placed in 37 DEG C, 5%CO248h or so is cultivated in incubator.Collect
Cell culture medium supernatant after transfection, centrifugation, cell fragment is removed, 0.45 μm of filter filtering, 10 times are concentrated with 30K super filter tube
Afterwards, it is sub-packed in 1.5mL centrifuge tubes, is saved backup in -80 DEG C of refrigerators.
B) preparation of vesicular stomatitis virus (VSV) pseudovirus
Using transfection reagent Lipofectamine 2000, by envelope plasmid pHEF-VSVG (by US National health research
Institute (NIH) aidsreagent projects provide) and skeleton plasmid pSG3 Δs env.cmvFLUC (referring to Nie J, et al.Sci
Rep.2017 Feb 20；7:42769.) mass ratio 1:2 ratio cotransfection 293T cells, 4-6h or so changes culture after transfection
Base, 37 DEG C are subsequently placed in, 5%CO248h or so is cultivated in incubator.The cell culture medium supernatant after transfection is collected, is centrifuged, is removed
Cell fragment, 0.45 μm of filter filtering, is sub-packed in 1.5mL centrifuge tubes, is saved backup in -80 DEG C of refrigerators.
1.3 pseudovirus titer determinations
A) pseudovirus frozen will be dispensed from -80 DEG C of refrigerators taking-up room-temperature water baths defrostings, it is dilute that 5 times of series are done in 96 orifice plates
Release, 11 gradients, 4 multiple holes, the μ L of final volume 100, last is classified as cell controls.
B) preprepared 293T cells (converging rate up to more than 80%) in incubator are taken out, by taking T75 blake bottles as an example,
The culture medium abandoned in bottle is inhaled, adds 5ml 1XPBS cleaning cells, after 1XPBS is abandoned in suction, adds 2.5ml has diluted 5 times 0.25%
Pancreas enzyme -EDTA, it is submerged cell dissociation 45 seconds, discard pancreatin, be placed in cell culture incubator and stand 2 minutes, added after 2min
In 10mlDMEM culture mediums and pancreatin, after piping and druming mixes, cell count, cell is diluted to 5 × 10 with DMEM complete mediums5
Individual/ml.The 100 μ L of addition per hole, i.e., 5 × 104/ hole, 96 orifice plates are put into incubator, cultivate 48h.
C) 96 orifice plates are taken out after 48h from cell culture incubator, is inhaled from sample well and abandons 100 μ L of supernatant, add 100 μ L balances
To the Bright-Glo Luciferase Assay Reagents of room temperature, lucifuge reaction 2min.
D) after reaction terminates, with multichannel pipettor by pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole, cell is made fully to split
Solution, suctions out 100 μ l liquid from every hole, is added in corresponding 96 hole chemiluminescence detection plates, is placed in chemiluminescence detector, uses
BrighGlo programs read luminous value.
E) with 3 times of the luminous value higher than cell control well for cut-off values, pseudovirus is calculated with Reed-Muench methods
TCID50, comprise the following steps that shown in：
1. calculate each viral dilution positive number of perforations (m) and negative number of perforations (n)；
2. calculate positive and negative hole cumulative number：Positive hole cumulative number from bottom to top add up (x), negative hole cumulative number by
It is upper to add up (y) downwards；
3. calculate the percentage in positive hole：Ratio=(x)/((x)+(y)) × 100；
4. calculate distance proportion：Distance proportion=(percent positive -50 for being more than 50%)/(big number is in 50% positive
Percentage-be less than 50% percent positive)；
5. logarithm+distance proportion × dilute of the highest extension rate of the percent positive of TCID50 logarithm=more than 50%
Release the logarithm of coefficient.
Evaluations of the embodiment 2.EBOV-10 to the inhibitory activity of Ebola's pseudovirus
In the present embodiment, by using Ebola's pseudovirus as described above to evaluate EBOV-10 (bentrl hydrothloride states
Family standard items, provided by National Institute for Food and Drugs Control's standard substance) Ebola virus inhibitory activity.
Because the pseudovirus skeleton plasmid pSG3 Δs env.cmvFluc that the present invention uses derives from HIV, therefore in order to exclude
Medicine suppresses the possibility of pseudovirus by preventing skeleton HIV process of reverse-transcription, and the present invention is utilizing Ebola's pseudovirus
While primary dcreening operation medicine, secondary screening is carried out to exclude vacation using pseudovirus is fitted together to comprising vesicular stomatitis virus (VSV) memebrane protein
It is positive.Calculate 50% inhibition concentration that medicine is fitted together to pseudovirus and the chimeric pseudovirus of Ebola virus memebrane protein to VSV memebrane proteins
(IC50) ratio, when the value is more than 3, it is possible to determine that exclude the medicine and pressed down by preventing skeleton HIV process of reverse-transcription
The possibility for virus of manufacturing the fake.Its specific experiment step is as follows：
A) using DMSO dissolvings EBOV-10 to 30mmol/L.
B) EBOV-10 is serially diluted in 96 orifice plates from 150 times of dilution startings, continuous 3 times of progress, 7 dilution factors, 2
Individual multiple holes, final volume are 100 μ L, and last first row and secondary series are respectively cell controls (only plus culture medium and cell) and virus
Control (only plus viral and cell).
C) Ebola's pseudovirus or VSV pseudovirus are diluted to 4000TCID50/ml respectively, 50 μ L are added per hole, i.e., it is every
Hole 200TCID50,96 orifice plates are put into 37 DEG C, 5%CO2Cultivate 1h.
D) when incubation time to half an hour, take out preprepared 293T cells in incubator (converge rate up to 80% with
On), pancreas enzyme treated cell is same as above.Cell is diluted to 5 × 10 with DMEM complete mediums5Individual/ml.
E) when incubation time is small to 1,100 μ l 5 × 10 are added per hole into 96 orifice plates5Individual/ml cell, make every hole thin
Born of the same parents are 5 × 104It is individual, 96 orifice plates are put into incubator, 37 DEG C, 5%CO2Cultivate 48h.
F) 96 orifice plates are taken out after 48h from cell culture incubator, is inhaled and abandoned on 150 μ l from each loading hole with multichannel pipettor
Clearly, 100 μ l Bright-GloTM Luciferase Assay Reagents, room temperature lucifuge reaction 2min are then added.
G) after reaction terminates, with multichannel pipettor by pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole, cell is made fully to split
Solution, suctions out 100 μ l liquid from every hole, is added in corresponding 96 hole chemiluminescence detection plates, is placed in chemiluminescence detector, uses
BrighGlo programs read luminous value.
H) inhibiting rate of calculating medicine, inhibiting rate=[(luminous intensity average-cell control well of sample sets is luminous strong by 1-
Spend average)/(virus control wells luminous intensity average-cell control well luminous intensity average)] × 100%；And with drug concentration
For abscissa, inhibiting rate is that ordinate draws amount effect curve.
I) according to the inhibiting rate curve of medicine, the ID50 and IC50 of following equation calculating medicine are utilized.
Medicine ID50=lg (1/ medicine is less than the dilution factor of 50% fluorescent value ratio)+(50- medicines are less than 50% fluorescent value
The fluorescent value percentage in ratio hole)/(the fluorescent value percentage for being higher than 50% fluorescent value ratio hole-it is less than 50% fluorescent value ratio
The fluorescent value percentage in hole) × lg (drug dilution multiple).
Medicine IC50 (μm ol/L)=30000/ medicine ID50.
The inhibiting rate curve of medicine is as shown in figure 1, by being calculated EBOV-10 to Ebola's pseudovirus (EBOV)
IC50 is 3.3 μM, and the IC50 to VSV pseudovirus is 44.4 μM, and both are noticeably greater than 3 by ratio.The above results show, EBOV-10
There is significant inhibitory action, and its combination by suppressing Ebola virus memebrane protein and target cell to Ebola's pseudovirus
So as to disturb infection of Ebola's pseudovirus to cell.
Embodiment 3.EBOV-10 Cytotoxic evaluation
In the present embodiment, quantitative determined by ATP with evaluate EBOV-10 medicines (bentrl hydrothloride national standard, by
National Institute for Food and Drugs Control's standard substance is provided) influence to cytoactive, its Cleaning Principle is as follows：
ATP is an index of living cells metabolism, is usedLuminescence method cell viability detects
Kit, number of viable cells, cell cracking in culture after medicine and cytosis are detected by being quantitative determined to ATP
It is directly proportional to existing ATP amounts with caused luminous signal, and ATP amounts are directly directly proportional to the cell quantity in culture.It is logical
Influence of the medicine to ATP luminous signals in cell is crossed, calculates 50% cytotoxic concentration (CC50) of medicine.Calculate medicine
Ratio (that is, therapeutic index SI=CC50/IC50) of 50% cytotoxic concentration (CC50) with medicine to the IC50 of target viral,
When the therapeutic index is more than 3, the medicine is judged to suppress the positive drug of Ebola virus, that is, it is notable to show that the medicine has
Suppression virus activity, and without obvious cytotoxicity.Its specific experiment step is as follows：
A) EBOV-10 medicines are serially diluted in 96 orifice plates from 150 times of dilution startings, continuous 3 times of progress, 7 dilutions
Degree, 2 multiple holes, final volume are 100 μ L, and last first row and secondary series are respectively that negative control (only adds culture medium, not refinement
Born of the same parents) and cell controls (only plus culture medium and cell).
B) cell controls and sample well add 50 μ L culture mediums per hole, 96 orifice plates are put into 37 DEG C, 5%CO2Cultivate 1h.
C) preprepared 293T cells (converging rate up to more than 80%) in incubator are taken out, pancreas enzyme treated cell is same
On.Cell is diluted to 5 × 10 with DMEM complete mediums5Individual/ml.100 μ l complete mediums, remaining hole are added in first row
100 μ l are added per hole and have been diluted to 5 × 105Individual/ml 293T cells, it is 5 × 10 to make every hole cell4It is individual, 96 orifice plates are put into and incubated
Case, 37 DEG C, 5%CO2Cultivate 48h.
D) 96 orifice plates are taken out after 48h from cell culture incubator, is inhaled and abandoned on 150 μ l from each loading hole with multichannel pipettor
Clearly, 100 μ l are then addedDetection reagent, room temperature lucifuge reaction 10min.
E) after reaction terminates, with multichannel pipettor by pressure-vaccum 3-5 times repeatedly of the liquid in reacting hole, cell is made fully to split
Solution, suctions out 100 μ l liquid from every hole, is added in the corresponding hole chemiluminescence detection plate of black 96, is placed in chemiluminescence detector
In, read luminous value with CellTiter-Glo programs.
F) influences (inhibiting rate) of the calculating EBOV-10 to cytoactive, inhibiting rate=[(luminous intensity of sample sets is equal by 1-
Value-negative control hole luminous intensity average)/(cell control well luminous intensity average-negative control hole luminous intensity average)] ×
100%；And using drug concentration as abscissa, inhibiting rate is that ordinate draws amount effect curve.
G) according to above-mentioned inhibiting rate curve, 50% cytotoxicity that the formula described in reference embodiment 2 calculates medicine is dense
Spend (CC50).
As a result as shown in Fig. 2 wherein EBOV-10 is 109 μM to the CC50 of cell, show that EBOV-10 is right below 109 μM
Cell does not have toxicity；Also, EBOV-10 CC50 values and its IC50 to Ebola virus ratio (that is, therapeutic index SI=
CC50/IC50 3) are noticeably greater than.The above results further demonstrate that EBOV-10 has significant suppression ebola disease cytotoxic activity, and
Without obvious cytotoxicity.
Evaluations of the embodiment 4.EBOV-10 to the neutralization activity of Ebola's live virus
In the present embodiment, using Ebola's live virus with evaluate EBOV-10 medicines (bentrl hydrothloride national standard,
There is provided by National Institute for Food and Drugs Control's standard substance) to the neutralization activity of Ebola virus, comprise the following steps that institute
Show：
A) be inoculated with Vero E6 cells (CRL-1586TM) into the orifice plate of black flat-bottom 96.
B) EBOV-10 that different diluted concentrations are added in respective aperture (proceeds by three times dilution from 17 μM, shares 7
Dilution factor, i.e., 0.023 μM~17 μM, dilution DMEM), virus control only adds the DMEM of same volume；At 37 DEG C, 5%CO2
Cell culture incubator in cultivate 1h.
C) Ebola's live virus strain EBOV/Mayinga-eGFP with eGFP autofluorescences is added (to be defended by Canada is public
Microbiological Lab of country of raw office professor Qiu Xiangguo gives；See, for example, Towner JS, et al.Virology.2005 Feb
5；332(1):20-7.), MOI is 0.1 TCID50/cell, is put into cell culture incubator and cultivates 1h.
D) remove supernatant, and add contain and b) in same concentrations medicine culture medium (DMEM/2%FBS), and be put into thin
72h is cultivated in born of the same parents' incubator.
E) each drug concentration sets 3 parallel holes.
F) after 72h, light levels are detected on BioTek Synergy HT plate reader.
G) method of reference embodiment 2, the inhibiting rate of medicine is calculated, and using drug concentration as abscissa, inhibiting rate is vertical
Coordinate draws amount effect curve；According to the inhibiting rate curve of medicine, calculate medicine medium effective concentration EC50 and 90% it is effective
Concentration EC90.
EBOV-10 inhibiting rate curve is as shown in figure 3, result shows that EBOV-10 has significantly to Ebola's live virus
Inhibitory activity.By the method for embodiment 2, medium effective concentrations (EC50) of the EBOV-10 to Ebola's live virus is calculated
For 3.3 μM, confidential interval is 2.3-4.7 μM, and 90% valid density (EC90) is 12.9 μM.The above results show, EBOV-10 pairs
Ebola's live virus has significant neutralization activity, can substantially suppress infection of Ebola's live virus to cell.
The evaluation of anti-ebola disease cytotoxic activity in embodiment 5.EBOV-10 bodies
In the present embodiment, using after Ebola virus is attacked BALB/c mouse as model, investigated EBOV-10 (hydrochloric acid
Bentyl national standard, provided by National Institute for Food and Drugs Control's standard substance) inside anti-Ebola virus live
Property.
4-5 week old female BAl BIc/c mouse (being provided by National Institute for Food and Drugs Control's Animal Resource Center) 20,
Divide 2 groups, every group 10.Experimental group：, for D0, to attack poison on the day of attacking poison a few days ago (Day-2), attack malicious the previous day (Day-1), attack
The bentrl hydrothloride (solvent is sterilized water) for giving 0.12mg/200 μ l is injected intraperitoneally in 4 hours (- H4) respectively before poison, and H0 attacks poison,
It is 60AID50 to attack toxic agent amount, and used virus is pHIV-ZGP-Fluc pseudovirus (angstrom prepared by embodiment 1.2
It is rich to draw pseudovirus)；Control group：Inject the sterilized water of same volume in the same way simultaneously with experimental group, H0 attacks poison, attacks toxic agent
Amount is identical with experimental group.The 4th day after poison is attacked, give every μ l yellow Jackets (240mg/kg) of mouse peritoneal injection 100 and
100 μ l animal luminous substrates, reaction carry out inspection light in living imaging instrument after 10 minutes, observe animal light levels.
As a result as shown in figure 4, compared with control group, the light levels of EBOV-10 administration groups are obvious weaker (Fig. 4 A),
The total light flux of EBOV-10 administration groups have dropped 64.66% (Fig. 4 B) compared with control group.The above results show, in Mice Body
In interior, EBOV-10 can substantially suppress the infection of Ebola virus, available for prevent and/or treat Ebola virus infection and
Disease caused by Ebola virus infects.
Embodiment 6.EBOV-10 and the evaluation of Ebola virus memebrane protein binding activity
In the present embodiment, surface plasma resonance (SPR) technical Analysis EBOV-10 (bentrl hydrothloride country marks are passed through
Quasi- product, provided by National Institute for Food and Drugs Control's standard substance) to the binding activity of Ebola virus memebrane protein.
With Ebola virus memebrane protein (being given by the high good fortune academician of the Chinese Academy of Sciences) for stationary phase, with different diluted concentrations
EBOV-10 be mobile phase (proceed by gradient dilution, respectively 200 μM from 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM,
6.25 μM, 3.125 μM, 1.5 μM).Buffer is PBS+0.05%Tween20+5%DMSO, at 25 DEG C, on BiacoreS200
It is measured.
As a result as shown in figure 5, wherein Fig. 5 A are the EBOV-10 of different diluted concentrations SPR；Fig. 5 B ring for stable state
The change curve relative to different EBOV-10 concentration should be worth, wherein vertical line represents equilibrium dissociation constant (KD) value, pass through
BiacoreS200 measure draws the K that EBOV-10 is combined with virus membrane antigenDFor 65 μM, show EBOV-10 and Ebola virus
Memebrane protein has certain binding activity.
Although the embodiment of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root
According to all teachings announced, various modifications and changes can be carried out to details, and these change in the guarantor of the present invention
Within the scope of shield.The whole of the present invention is divided into be provided by appended claims and its any equivalent.
Claims (8)
- Prevent and/or treat in subject 1. the compound or its pharmaceutically acceptable salt shown in logical formula (I) are used to prepare The purposes of the pharmaceutical composition of Ebola virus infection or the disease caused by Ebola virus infects；Wherein, m, x, y are each independently 0,1,2,3 or 4；Preferably, x is identical with y；Preferably, m, x, y are mutually the same；Preferably, x is identical with y, and is 0,1,2 or 3；Preferably, m, x, y are mutually the same, and are 0,1,2 or 3.
- 2. the purposes of claim 1, wherein, the compound has chemical formula as follows：
- 3. the purposes of claim 1 or 2, wherein, the disease caused by Ebola virus infects is Ebola hemorrhagic fever.
- 4. any one of claim 1-3 purposes, wherein, the pharmaceutically acceptable salt is hydrochloride, sulfate or phosphoric acid Salt.
- 5. any one of claim 1-4 purposes, wherein, described pharmaceutical composition includes prevention or the right of therapeutically effective amount will Seek the compound defined in 1 or 2 any one.
- 6. any one of claim 1-5 purposes, wherein, described pharmaceutical composition also includes can be used for treatment Ebola virus sense The other forms of pharmacologically active agents of dye and/or Ebola hemorrhagic fever, such as sertraline hydrochloride, clomiphene (Clomiphene), support Rui meter Fen (Toremifene), Favipiravir or NB-DNJ (Miglustat).
- 7. any one of claim 1-6 purposes, wherein, described pharmaceutical composition is tablet, pill, capsule, granule, dissipated Agent, solution, supensoid agent, parenteral solution or injection sterile powder.
- 8. any one of claim 1-7 purposes, wherein, the subject is mammal, such as non-human primate, example Such as people.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110028564A1 (en) * | 2009-02-20 | 2011-02-03 | Johansen Lisa M | Compositions and methods for treatment of filovirus-mediated diseases |
| CN103720684A (en) * | 2013-12-11 | 2014-04-16 | 武汉威立得生物医药有限公司 | Application of dicyclomine hydrochloride in preparation of drug for treating or preventing influenza virus infection |
| CN106573048A (en) * | 2014-08-04 | 2017-04-19 | 日东电工株式会社 | Composition for enhancing induction of humoral immunity, and vaccine pharmaceutical composition |
-
2017
- 2017-10-26 CN CN201711012931.3A patent/CN107789344A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110028564A1 (en) * | 2009-02-20 | 2011-02-03 | Johansen Lisa M | Compositions and methods for treatment of filovirus-mediated diseases |
| CN103720684A (en) * | 2013-12-11 | 2014-04-16 | 武汉威立得生物医药有限公司 | Application of dicyclomine hydrochloride in preparation of drug for treating or preventing influenza virus infection |
| CN106573048A (en) * | 2014-08-04 | 2017-04-19 | 日东电工株式会社 | Composition for enhancing induction of humoral immunity, and vaccine pharmaceutical composition |
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