CN106176700B - Application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament - Google Patents
Application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament Download PDFInfo
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- 239000003814 drug Substances 0.000 title claims abstract description 10
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
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- 108020005199 Dehydrogenases Proteins 0.000 description 1
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- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
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- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Pain & Pain Management (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament, niclosamidum is found through experiments that treated that the expression of intracellular KSHV or EBV related genes is substantially reduced in inventor, and the release of extracellular virion also decreased significantly.These inhibiting effect are in concentration dependent, and just have good antivirus action in the concentration of not overt toxicity, provide strong theoretical foundation and experimental basis for the research and development of further antiviral drugs, have important research and development value and development significance.Based on this, niclosamidum can be developed as safely and effectively anti-KSHV or EBV drugs.
Description
Technical field
The present invention relates to a kind of new opplication of compound, more particularly to niclosamidum is preparing anti-tumorigenesis herpesvirus medicament
In application.
Background technology
Kaposi's sarcoma associated herpesvirus KSHV（Kaposi's sarcoma-associated herpesvirus）It is
A member in herpes virus group.KSHV infects the malignant tumour that the Kaposi's sarcoma KS induced is common in AIDS patient,
About 20% AIDS patients can be with Kaposi's sarcoma, and AIDS-KS patient death rates are high, and survival rate only has within 5 years
About 8%.95% or more individual does not show clinical symptoms and illness after KSHV infection in normal population.But in immunosuppressive trouble
Person, in AIDS patient, Organ Transplantation Patients and chemicotherapy patient, KSHV has very high infection rate and greatly harm, can
Lead to Kaposi's sarcoma（Kaposi ' s sarcoma, KS）, primary effusion lymphoma（primary effusion
Lymphoma, PEL）And multicenter Karst is sick slowly（Multicentric castleman disease, MCD）Etc. diseases.
In recent years in other diseases（Such as HBV chronic hepatitis, drug abuse and geriatric disease）It has also been found that very high KSHV infection rates and evil
Property tumour, just gradually draws attention.
Epstein-Barr virus（Epstein-barr virus, EBV）γ-herpesviral is belonged to KSHV, EBV can be passed by saliva
It broadcasts, and researches show that this viruses can also be propagated by sexual behaviour in recent years some.EBV infects the whole world 5 years old or more at present
95% or more individual in crowd, after infection, EBV is present in throughout one's life in carrier's body, is in latent infection state, can cause more
Kind human diseases, including infectious mononucleosis（Infection Mononucleosis, IM）, oral hairy leukoplakia
Disease, the nervous system disease multiple sclerosis（Multiple Sclerosis, MS）, Gianotti-Crosti syndromes, multiform
Erythema（Erythema Multiforme, EM）, lymphadenia multiple after Lipschutz ulcer and solid organ transplantation
Property disease.Currently used inhibition viral DNA synthesizes to treat the drug of herpesvirus infection（Such as acyclovir and its correlationization
Close object）Poor to both virus infection curative effects, vaccine is also still in development phase.
Niclosamidum is approved have research to send out in addition as a kind of anthelmintic, safety by the World Health Organization
Existing niclosamidum may have certain effect to the treatment of bacterium infection.There is not specific experimental data to show niclosamidum to KSHV
There is the report of pharmaceutical activity with EBV.
Invention content
The purpose of the present invention is to provide application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament.
Inventor is with the technologies such as Western Bolt and Real-time quantitative PCR, detection KSHV sun
The expression of KSHV related genes and the content of extracellular virion in property cell BCBL1；EBV positive cells are detected, including
The expressing of EBV related genes, the content of extracellular virion in lymphocyte P3HR-1 and epithelial cell HNE1-2089.
Experiment finds niclosamidum treated that the expression of intracellular KSHV or EBV related genes is substantially reduced, and extracellular virus
Particle release also decreased significantly.These inhibiting effect are in concentration dependent, and are just had very well in the concentration of not overt toxicity
Antivirus action, for further antiviral drugs research and development strong theoretical foundation and experimental basis are provided, have it is important
Research and development value and development significance.Based on this, niclosamidum can be developed as safely and effectively anti-KSHV or EBV drugs.
Description of the drawings
Fig. 1：Niclosamidum is in BCBL1 into the cell to KSHV incubation period albumen LANA, burst times albumen RTA, K8, ORF64
Expression influence；
Fig. 2：Niclosamidum is in the P3HR-1 effects to EBV GAP-associated protein GAPs EA-D, ZTA expression into the cell；
Fig. 3：Niclosamidum is in the HNE1-2089 effects to EBV GAP-associated protein GAPs EA-D, ZTA expression into the cell；
Fig. 4：The influence of the niclosamidum viral yield intracellular to BCBL1；
Fig. 5：Influence of the niclosamidum to the extracellular viral yields of P3HR-1；
Fig. 6：Influence of the niclosamidum to the extracellular viral yields of HNE-2089；
Fig. 7：Survival Effects of the niclosamidum to PBMC cells.
Specific implementation mode
With reference to experiment, the technical solution that further illustrates the present invention.
In testing below, unless stated otherwise, reagent that the present invention uses, device and method are routinely adopted for the art
Reagent, equipment and the conventional use of method of purchase.
Experimental principle：As γ-herpesviral, life cycle is all divided into incubation period and burst times, cell quilt by EBV, KSHV
Incubation period is quickly entered after infection after of short duration acute reaction, and passes through special stimulation, cell enters burst times progress
Virus amplification is bred.Therefore, the correlative protein expression for detecting incubation period and burst times can be used as assessment drug for two kinds of diseases
A kind of means of toxic action.
It is abbreviated as in the present invention：TPA, euphorbia diterpenoids ester；NaB, sodium butyrate
Experiment 1：In the BCBL1 expression to KSHV incubation period albumen LANA, burst times albumen RTA, K8, ORF64 into the cell
Influence
1) well-grown BCBL1 cells are taken, are inoculated in 6 hole clear flat bottom plates, per hole 2 × 106Cell.The training used
Foster base is complete medium：RPMI1640,10% fetal calf serum and 1% dual anti-, condition of culture be 5% carbon dioxide, 37 DEG C；
2) derivant TPA is added after 12h, final concentration distinguishes 20ng/mL；
3) niclosamidum of gradient concentration is added after 3 hours, final concentration is respectively 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μ
M；
4) after cultivating 48h, 1000rpm, 10min harvest cell, carry out protein blot experiment.
As a result as Fig. 1 is shown：It is dense by niclosamidum in the expression of intracellular KSHV GAP-associated protein GAPs RTA, K8, the ORF64 of BCBL1
The influence of degree is in certain concentration dependent.
Experiment 2：Niclosamidum is in the P3HR-1 effects to EBV GAP-associated protein GAPs EA-D, ZTA expression into the cell
1) well-grown P3HR-1 cells are taken, are inoculated in 6 hole clear flat bottom plates, per hole 2 × 106Cell.It uses
Culture medium is complete medium：RPMI1640,10% fetal calf serum and 1% dual anti-, condition of culture be 5% carbon dioxide, 37 DEG C；
2) derivant TPA (final concentration of 20 μ g/ml), NaB is added after passing on 12h（Final concentration of 3mM）；
3) niclosamidum is added after inducing 3 hours, final concentration is respectively 0 μM, 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μ
M,2μM,5μM；
4) it after the 48h after derivant is added, collects cell and carries out protein blot experiment.
As a result as Fig. 2 is shown：Intracellular in P3HR-1, the expression of EBV GAP-associated protein GAPs ZTA, EA-D are by niclosamidum concentration
Influence, be in certain concentration dependent.
Experiment 3：Niclosamidum is in the HNE1-2089 effects to EBV GAP-associated protein GAPs EA-D, ZTA expression into the cell
1) well-grown HNE1-2089 cells are taken, 1：4 are inoculated in 6 hole clear flat bottom plates；
2) 12h cells are adherent, and derivant TPA (final concentration of 20 μ g/ml), NaB is added（Final concentration of 3mM）
3) niclosamidum of gradient concentration is added after 3h, final concentration is respectively 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μ
M,5μM；
4) after cultivating 48h, cell is scraped in plate with cell scraper plate and is taken out, cold PBS rinsings, 1000rpm, 10min,
4 DEG C are collected by centrifugation cell and carry out protein blot experiment.
Experimental result is as shown in Figure 3：Intracellular EBV GAP-associated protein GAPs ZTA, the EA-D of HNE1-2089 expression by chlorine nitre willow
The influence of amine concentration is in certain concentration dependent.
Experiment 4：The influence of the niclosamidum viral yield intracellular to BCBL1
1) well-grown cell line BCBL1 is taken, is planted into 48 orifice plates, cell dosage is 1 × 105/ hole, cell is divided into
Do not induce group（3 holes）With induction group（21 holes）；
2) derivant TPA, final concentration of 20ng/mL is added in induction group after 3h；
3) niclosamidum of respective concentration is added after 3h, concentration distinguishes 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μ
M, 3 holes of each concentration；
4) cell is harvested after 120h, 10000g, 4 DEG C take supernatant；
5) extracellular virus DNA is extracted, method is as follows：
A) it takes 200 μ L supernatants, 2 μ L DNase I is added, 20 μ L10 × DNase I buffer are incubated in 37 DEG C of incubators
Educate 1h；
B) 10 μ L of 0.5M EDTA are added into aforesaid liquid, mixing terminates reaction；
C) 80 DEG C of water-baths inactivate 10min；
D) 20 μ L proteinase K solution are added, 200 μ L AL buffer, vortex mixing, 56 DEG C of water are and then added
Bathe 10min；
E) DNA in 440 μ L phenol chloroform virions, vortex mixing is added；
F) 12000rpm, 4 DEG C of centrifugation 10min；
G) it is layered after centrifuging, about 300 μ L of supernatant is taken, until in another ep pipes.Avoid the phenol chloroform for being extracted into lower layer；
H) 6 μ L of 5M NaCl solutions are added, 750 μ L of absolute ethyl alcohol are added, are eventually adding 1 μ L of glycogen；
I) it is put into -80 DEG C of refrigerators after mixing well, places 1h；
J) sample is taken out, 12000rpm, 4 DEG C from refrigerator, supernatant is removed after 30min centrifugations；
K) there are DNA precipitations in bottom in Ep pipes, is washed with 1mL70% ice ethyl alcohol, closes the lid, turn upside down several times；
L) 12000rpm, 4 DEG C of centrifugation 10min, abandons supernatant；
M) 12000rpm, 4 DEG C of centrifugation 10min, removes remaining alcohol；
N) lid is opened, extra alcohol is allowed to volatilize；
O) 40 μ L ddH are added after 3min2O dissolving DNAs；
6) Real-time quantitative PCR detect viral level.
The results are shown in Figure 4.The results show that niclosamidum is in concentration dependant in the extracellular yield of BCBL1 for KSHV
Property inhibit.
Experiment 5：Influence of the niclosamidum to the extracellular viral yields of P3HR-1；
1) well-grown cell line P3HR-1 is taken, is planted into 48 orifice plates, cell dosage is 1 × 105/ hole, by cell point
Not induce group and induction group；
2) induction group is added after 3h and derivant TPA, NaB is added, final concentration is respectively 20ng/mL, 3mM；
3) niclosamidum of respective concentration is added after induction 3h, concentration distinguishes 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μ
M, 5 μM of each 3 multiple holes of concentration；
4) cell is harvested after 120h, 10000g is centrifuged 10 minutes, and 4 DEG C take supernatant；
5) viral DNA is extracted, method is as follows：
A) A. takes 200 μ L supernatants, and 2 μ L DNase I, 20 μ L10 × DNase I buffer, in 37 DEG C of incubators are added
It is incubated 1h；
B) 10 μ L of 0.5M EDTA are added into aforesaid liquid, mixing terminates reaction；
C) 80 DEG C of water-baths inactivate 10min；
D) 20 μ L proteinase K solution are added, 200 μ L AL buffer, vortex mixing, 56 DEG C of water are and then added
Bathe 10min；
E) DNA in 440 μ L phenol chloroform virions, vortex mixing is added；
F) 12000rpm, 4 DEG C of centrifugation 10min；
G) it is layered after centrifuging, about 300 μ L of supernatant is taken, until in another ep pipes.Avoid the phenol chloroform for being extracted into lower layer；
H) 6 μ L of 5M NaCl solutions are added, 750 μ L of absolute ethyl alcohol are added, are eventually adding 1 μ L of glycogen；
I) it is put into -80 DEG C of refrigerators after mixing well, places 1h；
J) sample is taken out, 12000rpm, 4 DEG C from refrigerator, supernatant is removed after 30min centrifugations；
K) there are DNA precipitations in bottom in Ep pipes, is washed with 1mL70% ice ethyl alcohol, closes the lid, turn upside down several times；
L) 12000rpm, 4 DEG C of centrifugation 10min, abandons supernatant；
M) 12000rpm, 4 DEG C of centrifugation 10min, removes remaining alcohol；
N) lid is opened, extra alcohol is allowed to volatilize；
O) 40 μ L ddH are added after 3min2O dissolving DNAs；
6) Real-time quantitative PCR detect viral level.
The results are shown in Figure 5.The results show that niclosamidum can inhibit the extracellular viruses of EBV to discharge EBV, and it is in
Concentration dependent inhibits.
Experiment 6：Influence of the niclosamidum to the extracellular viral yields of HNE-2089；
1) well-grown cell line HNE1-2089 is taken, is planted into 24 orifice plates, cell dosage is 2 × 105/ hole, by cell
It is divided into and does not induce group and induction group；
2) cell is adherent after 12h, derivant TPA, NaB is added, final concentration is respectively 20ng/mL, 3mM；
3) niclosamidum of respective concentration is added after induction 3h, concentration distinguishes 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μ
M, 5 μM of each 3 multiple holes of concentration；
4) cell is harvested after 120h, 10000g is centrifuged 10 minutes, and 4 DEG C take supernatant；
5) viral DNA is extracted, method is as follows：
A) it takes 200 μ L supernatants, 2 μ L DNase I is added, 20 μ L10 × DNase I buffer are incubated in 37 DEG C of incubators
Educate 1h；
B) 10 μ L of 0.5M EDTA are added into aforesaid liquid, mixing terminates reaction；
C) 80 DEG C of water-baths inactivate 10min；
D) 20 μ L proteinase K solution are added, 200 μ L AL buffer, vortex mixing, 56 DEG C of water are and then added
Bathe 10min；
E) DNA in 440 μ L phenol chloroform virions, vortex mixing is added；
F) 12000rpm, 4 DEG C of centrifugation 10min；
G) it is layered after centrifuging, about 300 μ L of supernatant is taken, until in another ep pipes.Avoid the phenol chloroform for being extracted into lower layer；
H) 6 μ L of 5M NaCl solutions are added, 750 μ L of absolute ethyl alcohol are added, are eventually adding 1 μ L of glycogen；
I) it is put into -80 DEG C of refrigerators after mixing well, places 1h；
J) sample is taken out, 12000rpm, 4 DEG C from refrigerator, supernatant is removed after 30min centrifugations；
K) there are DNA precipitations in bottom in Ep pipes, is washed with 1mL70% ice ethyl alcohol, closes the lid, turn upside down several times；
L) 12000rpm, 4 DEG C of centrifugation 10min, abandons supernatant；
M) 12000rpm, 4 DEG C of centrifugation 10min, removes remaining alcohol；
N) lid is opened, extra alcohol is allowed to volatilize；
O) 40 μ L ddH are added after 3min2O dissolving DNAs
6) Real-time quantitative PCR detect viral level.
The results are shown in Figure 6.The results show that niclosamidum can inhibit EBV extracellular the epithelial cell of infection EBV
Viral yield, and in concentration dependent inhibit.
Experiment 7：Survival Effects of the niclosamidum to PBMC cells
MTS(3-(4,5-dimethylthiazol-2-yl)-5(3-carboymethoyphenyl)-2-(4-
Sulfopheny) -2H-tetrazolium, inner salt) it is a kind of newly synthesized tetrazole compound, it can be by living cells
A variety of dehydrogenases in mitochondria are reduced into respectively coloured first a ceremonial jade-ladle, used in libation product, and shade and the work of certain sensitive cells strains are thin
Born of the same parents' number is in highly relevant in a certain range.According to the absorbance value of the 490n measured（OD values）, to judge living cells quantity, OD
Value is bigger, and cell activity is stronger, then it represents that drug toxicity is smaller.Specific experiment operation is as follows：
1) inoculating cell, with the RPMI culture solutions containing 10% fetal calf serum by human peripheral blood mononuclear cell PBMC with every hole
10000 cell inoculations are to 96 orifice plates, per pore volume 100ul；
2) be added niclosamidum after 12h, final concentration is respectively 0 μM, 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM,
5μM ,10μM ,20μM,40μM,80μM；
3) after cultivating 48h, MTS solution 20ul are added per hole, continue to be incubated 2 ~ 4 h in the incubator；
4) 490nm wavelength is selected, each hole absorbance value is measured on enzyme linked immunological monitor, observation compound is thin to PBMC
The cytotoxicity of born of the same parents.
Experimental result as shown in fig. 7, the experimental results showed that, CC50>80 μM, antiviral compound toxicity is relatively low, in PBMC
Acellular poison phenomenon is rendered into cell.
The experimental results showed that the expression of niclosamidum treated intracellular KSHV or EBV related genes is substantially reduced, and
Extracellular virion release also decreased significantly.These inhibiting effect are in concentration dependent, and in not overt toxicity
Concentration just has good antivirus action, for further antiviral drugs research and development provide strong theoretical foundation and experiment according to
According to important research and development value and development significance.
Therefore, niclosamidum can be by the duplication and expression of inhibition KSHV and EBV, to disease caused by KSHV and EBV
Preferable therapeutic effect is played, especially to Kaposi's sarcoma caused by KSHV, primary effusion lymphoma, multicenter Karst
Slow disease has preferable prevention and therapeutic effect.
Niclosamidum can be used with the active ingredient combination of other anti-tumorigenesis herpesvirals, be inhibited through a variety of ways
The duplication and expression of KSHV and EBV to Kaposi's sarcoma caused by disease, especially KSHV caused by KSHV and EBV, primary is oozed
Going out property lymthoma, the slow disease in multicenter Karst play better therapeutic effect.
Claims (1)
1. application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament, wherein the tumorigenesis herpesviral is EBV.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022147499A1 (en) * | 2021-01-04 | 2022-07-07 | Neurobo Pharmaceuticals, Inc. | Method of treating viral infections with a combination of niclosamide and gemcabene |
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| CN111569798B (en) * | 2020-05-27 | 2021-08-17 | 中山大学 | Degradable core-shell calcium alginate oxide gel microspheres and preparation method and application thereof |
| CN117281800A (en) * | 2023-11-10 | 2023-12-26 | 重庆医科大学 | Application of niclosamide in preparing medicine for treating hepatitis B |
Citations (1)
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|---|---|---|---|---|
| CN1120389A (en) * | 1995-05-30 | 1996-04-17 | 南京药物研究所 | Synergistic compound for killing snail |
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2016
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120389A (en) * | 1995-05-30 | 1996-04-17 | 南京药物研究所 | Synergistic compound for killing snail |
Non-Patent Citations (4)
| Title |
|---|
| Epidemiology, Pathophysiology, and Treatment of Kaposi Sarcoma-Associated Herpesvirus Disease: Kaposi Sarcoma, Primary Effusion Lymphoma, and Multicentric Castleman Disease;Ryan J. Sullivan等;《Clinical Infectious Diseases》;20080922;第47卷;第1209-1215页 * |
| Identification of Niclosamide as a New Small-Molecule Inhibitor of the STAT3 Signaling Pathway;Xiaomei Ren等;《ACS Med. Chem. Lett.》;20100907;第1卷;第454-459页 * |
| Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma;Yoshiyasu Aoki等;《BLOOD》;20030215;第101卷(第4期);第1535-1542页 * |
| Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects;Andreas Jurgeit等;《PLOS Pathogens》;20121025;第8卷(第9期);第1-14页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022147499A1 (en) * | 2021-01-04 | 2022-07-07 | Neurobo Pharmaceuticals, Inc. | Method of treating viral infections with a combination of niclosamide and gemcabene |
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