CN102755316A - Application of artemisinin and derivatives thereof in preparation of medicaments for treating hepatitis C viruses - Google Patents
Application of artemisinin and derivatives thereof in preparation of medicaments for treating hepatitis C viruses Download PDFInfo
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- CN102755316A CN102755316A CN2012102433696A CN201210243369A CN102755316A CN 102755316 A CN102755316 A CN 102755316A CN 2012102433696 A CN2012102433696 A CN 2012102433696A CN 201210243369 A CN201210243369 A CN 201210243369A CN 102755316 A CN102755316 A CN 102755316A
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Abstract
The invention discloses an application of artemisinin and derivatives thereof in preparation of medicaments for treating hepatitis C viruses (HCV). The medicaments which almost do not have toxic concentration are used for anti-virus test; and by detecting the influence of the artemisinin and the derivatives thereof such as dicyanogen artemisinin, artemether and artesunate on HCV replication, the artemisinin and the derivatives thereof serving as small molecular compounds have remarkable viral activity resistance and are dose-dependent. The artemisinin and the derivatives thereof which have already been used for clinical antimalarials have low toxic or side effect on a human body. The artemisinin and the derivatives thereof which almost do not have toxicity can inhibit about 90 percent of HCV replication, and are efficient and low-toxic anti-HCV compounds. The compounds have a broad prospect for developing anti-HCV medicaments.
Description
Technical field
The present invention relates to belong to medical technical field, more specifically relate to the application in preparation treatment hepatitis C virus (HCV) medicine of a kind of arteannuin and derivant thereof.
Background technology
(Hepatitis C Virus HCV) is found in 1989 to hepatitis C virus, and it is the main pathogens that causes NANB-PTH.The whole world has 1.7 hundred million people's HCV infection approximately at present.HCV infects and is worldwide distribution, and infection rate has 3% approximately, wherein annual newly-increased case 300～4,000,000.China's seroepidemiological survey data shows that general crowd HCV positive rate is 3.2%.The main route of transmission of HCV comprises propagation, IDU propagation, property contact and the mother-to-baby transmission etc. of blood transfusion or blood product.HCV infects and very easily causes chronicity, but has only the HCV the infected's spontaneous recovery about 20%, and about actute infection person of 80% can be developed into chronic infection, and wherein 20% chronic infection person can develop into liver cirrhosis even hepatocarcinoma at 5 to 10 end of the year thereafter.HCV also can cause the pathological changes of its hetero-organization and organ except meeting influences liver.For example: mixed type condensation globulinemia, non_hodgkin lymphoma and film property hypertrophy property acute glomerulonephritis.
Present stage, the Therapeutic Method of anti-HCV mainly was confined to the combined therapy of the long-acting IFN-(peg-IFN) and the virazole (Ribavirin) of polyethylene glycol conjugation.This therapy has only 40% (1 type HCV the infected)～80% (2 type HCV the infected) to the cure rate of HCV, and the course of treatment is long, costs an arm and a leg, and side effect is obvious.Certainly, along with to the deepening continuously of HCV research, many is that the medicine of target spot continues to bring out with HCV albumen.Telaprevir and Boceprevir belong to protease inhibitor in the direct antiviral drugs (PI); Along with these two direct antiviral drugs Telaprevir and the Boceprevir listing successively in 2011, the standard care scheme of hepatitis C will have great improvement.But still not having effective vaccine prevention HCV at present infects.Therefore, HCV infects an important public hygiene problem that remains present harm whole world population health.
Arteannuin (Artemisinin) be from feverfew Hemerocallis citrina Baroni mugwort extraction separation to a kind of sesquiterpene lipoid chemical compound with peroxide bridge; Multiple derivant such as dihydroarremisine (dihydroartemisinin) have been developed on this basis again; Artemether (Artemether), artesunate (artesunate) etc.Arteannuin and derivant thereof all have malaria active, and be effective to various malaria.Still there are not at present arteannuin and the application of derivant aspect anti-hepatitis c virus thereof.
Summary of the invention
The objective of the invention is to remedy the deficiency of prior art; Be to be to provide a kind of micromolecular compound arteannuin and the application of derivant in preparation treatment hepatitis C virus cytotoxic drug thereof, thereby be that therapy for hepatitis C provides one type of micromolecular compound that safe and efficient toxic and side effects is little clinically.Arteannuin and derivant thereof can suppress duplicating of hepatitis C virus effectively in the avirulence scope, can further be developed as the medicine of treatment infection with hepatitis C virus disease, are with a wide range of applications.
In order to realize above-mentioned purpose, the technical scheme that the present invention adopts is:
The application in preparation treatment hepatitis C virus cytotoxic drug of a kind of chemical compound arteannuin and derivant thereof, the structure and the molecular formula of this compounds are respectively:
Structure formula I arteannuin structure formula II dihydroarremisine
Structure formula III Artemether structure formula IV artesunate
Wherein: H is a hydrogen atom; O is an oxygen atom; OH is a hydroxyl; CH3 is a methyl; OCH3 is a methoxyl group; COOH is a carboxyl.
The application in preparation treatment hepatitis C virus (HCV) medicine of a kind of arteannuin and derivant thereof the steps include:
A. arteannuin and derivant thereof are to the cytotoxicity experiment of Huh7.5.1: the Huh7.5.1 cell is by 8 * 10
3Individual cells/well is inoculated in the 96 porocyte culture plates, behind the cell attachment, handles with the medicine of different gradient concentrations respectively, and every group is repeated three holes, place 37 ℃, 5%CO
2Cultivate in the incubator after 24 hours, with 3-(4,5-dimethylthiazole-2)-2, (tetrazolium bromide, MTT) method detects the survival rate of cell to 5-diphenyl tetrazole bromine salt.
B. arteannuin and the active evaluation of derivant anti-hepatitis c virus thereof: the structure of (1) plasmid PJFH1-5AGFP: on PJFH1, carry out the plasmid transformation; (nt7523 ~ nt7528, aa419 ~ aa420) insert the EGFP gene to choose restriction enzyme site XhoI of NS5A coding region C end.(2) preparation of viral JFH1-5AGFP: the plasmid PJFH1-5AGFP with after the XbaI linearisation is a template, and in vitro transcription obtains virus genome RNA, and electricity changes the Huh-7.5.1 cell then.After 9～10 days, obvious cytopathy will appear, then collect supernatant of culture medium, after the packing-80 ℃ frozen.In order to obtain a large amount of viral storage liquid, the infection multiplicity with 0.02 is with viral infection Huh7.5.1 cell, wait to occur obvious cytopathy after, collect infectious supernatant, store for use.(3) the Huh7.5.1 cell is pressed 8 * 10
3Individual/hole is inoculated in the 96 porocyte culture plates, cultivate 14～18h in 37 ℃ of cell culture incubators after, it is subsequent use to treat that cell grows up to behind the monolayer.With the DMEM culture medium arteannuin and derivant thereof are diluted to 8 Concentraton gradient for 2 times, every group of 2 repetitions.The various dose medicine is joined in the culture dish behind the 12h, and the infection multiplicity by 0.2 adds JFH1-5AGFP virus, infects back 72h and carries out the GFP fluorescence signal and detect.Study the medicine antiviral activity through observing NS5A-GFP fluorescence intensity variation in situ detection virus N S5A-GFP gene expression then.
The derivant of described arteannuin comprises dihydroarremisine, Artemether, and artesunate, arteether is in the derivant of all interior arteannuin.
Described anti-hepatitis C medicine is as active constituents of medicine with the arteannuin or derivatives thereof; Utilize modern common drug preparation means, can arteannuin and derivant thereof be made active component and process tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, nanometer formulation, any pharmaceutically acceptable dosage form of injection.
The present invention compared with prior art has the following advantages and effect:
1. arteannuin and derivant thereof are to be used for clinical anti-malaria medicaments, explain that its toxic and side effects to human body is little.
2. arteannuin and derivant thereof be under avirulent basically condition, and HCV is duplicated 90% the inhibition of having an appointment, and is the anti-HCV chemical compound of one type of high-efficiency low-toxicity.
Description of drawings
Fig. 1 is the chemical structural formula of a kind of arteannuin and derivant thereof.
Chinese name: arteannuin
English name: Artemisinin
Molecular formula: C15H22O5
Molecular weight: 282.33
CAS number: 63968-64-9
Chinese name: dihydroarremisine
English name: dihydroartemisinin
Molecular formula: C15H24O5
Molecular weight: 284.35
CAS number: 81496-81-3
Chinese name: Artemether
English name: Artemether
Molecular formula: C16H26O5
Molecular weight: 298.37
CAS number: 71963-77-4
Chinese name: artesunate
English name: Artesunate
Molecular formula: C19H28O8
Molecular weight: 384.42
CAS number: 88495-63-0
To be a kind of arteannuin and derivant thereof detect sketch map to the cytotoxicity of Huh7.5.1 to Fig. 2.
Figure detects arteannuin with mtt assay, dihydroarremisine, and Artemether, artesunate is to the Huh7.5.1 cytotoxicity.
Fig. 3 is a kind of Huh7.5.1 cell sketch map of handling the viral JFH1-5AGFP infection that is had reporter gene with arteannuin and derivant thereof.
A. after handling with arteannuin, study the medicine antiviral activity through observing NS5A-GFP fluorescence intensity variation in situ detection virus N S5A gene expression.
B. after handling with dihydroarremisine, study the medicine antiviral activity through observing NS5A-GFP fluorescence intensity variation in situ detection virus N S5A gene expression.
C. after handling with Artemether, study the medicine antiviral activity through observing NS5A-GFP fluorescence intensity variation in situ detection virus N S5A gene expression.
D. after handling with artesunate, study the medicine antiviral activity through observing NS5A-GFP fluorescence intensity variation in situ detection virus N S5A gene expression.
The specific embodiment
In order to understand content of the present invention better, below in conjunction with the practical implementation method content of the present invention is described further, but protection content of the present invention is not limited to following examples.
Embodiment 1: arteannuin and the active evaluation of derivant anti-hepatitis c virus thereof
The application in preparation treatment hepatitis C virus (HCV) medicine of a kind of arteannuin and derivant thereof the steps include:
1. experiment material
1.1 cell, plasmid, virus and medicine
The Huh7.5.1 cell is so kind as to give (asking for an interview the genetic resources table) by Dr.F.V.Chisari; The plasmid pJFH1 that contains HCV 2a type JFH1 Strain genom sequence is so kind as to give (asking for an interview the genetic resources table) by Dr.Takaji professor Wakita.The viral JFH1-5AGFP that has reporter gene is by this prepared in laboratory; Arteannuin, dihydroarremisine, Artemether, artesunate is available from sigma company.
1.2 reagent
The DMEM culture medium is available from GIBCO company; The MTT detectable is available from biomol company; Restricted enzyme is available from NEB company.
1.3 experimental apparatus
The senior inverted microscope Axio of Zeiss observer A1 (Carl Zeiss); The PerkinElmer multi-tester.
2. experimental technique and result
2.1 drug cell toxicity detects
37 ℃, cultivate in the 5%CO2 humidification incubator.Use contains the DMEM culture medium of penicillin and the streptomycin of 10%FBS, 100U/mL.Go down to posterity ratio 1/4 – 1/6 behind cell to 90% degree of converging.The Huh7.5.1 cell is by 8 * 10
3Individual cells/well is inoculated in the 96 porocyte culture plates, and is subsequent use behind the cell attachment; Use culture medium that medicine is carried out gradient dilution with 10mM as initial concentration, 3 multiple holes of every gradient.In every hole, add 5mg/ml MTT20 μ l after cultivating 72h, put and continue in the cell culture incubator to cultivate; After cultivating 4h, abandon the culture fluid supernatant, every hole adds 100 μ l/ holes, three lysates, and (lysate is by SDS10g; Isobutanol 5ml; 10M HCl 0.1ml is made into 100ml with the distilled water dissolving), multi-tester detected 570nm wavelength light absorption value after dissolving was spent the night in 37 ℃ of incubators; Tuning wavelength is 630nm, and calculates each drug level cell survival rate.The result is as shown in Figure 2.The median lethal concentration to the Huh7.5.1 cell of all cpds is as shown in the table.
2.2 the structure of plasmid PJFH1-5AGFP and the preparation of viral JFH1-5AGFP;
On PJFH1, carry out the plasmid transformation, (nt7523 ~ nt7528, aa419 ~ aa420) insert EGFP gene (amplification is from the PEGFP-N1 plasmid) to choose restriction enzyme site XhoI of NS5A coding region C end.After the plasmid construction success, order-checking is identified.Plasmid PJFH1-5AGFP with after the XbaI linearisation is a template, and in vitro transcription obtains virus genome RNA, and electricity changes the Huh-7.5.1 cell then.After 9～10 days, obvious cytopathy will appear, then collect supernatant of culture medium, after the packing-80 ℃ frozen.In order to obtain a large amount of viral storage liquid, the infection multiplicity with 0.02 is with viral infection Huh7.5.1 cell, wait to occur obvious cytopathy after, collect infectious supernatant, store for use.
Come the disease-resistant strain JFH1-Luc-5AGFP of in situ detection T0901317 active 2.3 observe the GFP positive rate;
The viral JFH1-5AGFP that has reporter gene has the GFP fluorescence labels at HCV non-structural protein NS5A C-terminal, can with fluorescence microscope in living cells Real Time Observation HCV in cell infection and duplicate situation.The Huh7.5.1 cell is pressed 8 * 10
3Individual/hole is inoculated in the 24 porocyte culture plates, cultivate 14～18h in 37 ℃ of cell culture incubators after, it is subsequent use to treat that cell grows up to behind the monolayer.With the DMEM culture medium arteannuin and derivant thereof are diluted to 8 Concentraton gradient for 2 times, every group of 2 repetitions join in the culture dish, and behind the 12h, the infection multiplicity by 0.2 adds JFH1-5AGFP, infect back 72h and carry out the detection of GFP fluorescence signal.For NS5A-GFP is calibrated and observed to GFP fluorescence signal and cell number in intracellular inferior location, the applicant carries out nucleus dyeing experiment with DAPI.At first abandon the culture fluid supernatant, with fixative with cell at the fixing 30min of room temperature; Reuse PBS (pH7.4) washes 3 times, each 5min; Then add the DAPI dye liquor incubated at room 30min that combines the liquid dilution; Use PBS (pH7.4) to wash at last 3 times, each 5min; Under common inverted fluorescence microscope, observe fluorescence signal then.The GFP positive cell photo that fluorescence microscope is taken down is representative one group that from the photo in a plurality of different visuals field, chooses.
The result is as shown in Figure 3.Arteannuin concentration is at 15.63M, during 125M, the suppression ratio of HCV is about 80%, 90%, and the survival rate of Huh7.5.1 cell is about 99%, 79%.Dihydroarremisine concentration is at 2.5M, during 5M, the suppression ratio of HCV is about 95%, 98%, and the survival rate of Huh7.5.1 cell is about 100%, 81%.Artemether concentration is at 31.25M, during 62.5M, the suppression ratio of HCV is about 85%, 90%, and the survival rate of Huh7.5.1 cell is about 98%, 98%.The concentration of artesunate is 2.5M, during 5M, the suppression ratio of HCV is about 95%, 99%, and the survival rate of Huh7.5.1 cell is about 95%, 81%.In sum, arteannuin and derivant thereof have all shown good anti-HCV activity.
Claims (3)
1. arteannuin and derivant thereof the application in preparation treatment hepatitis C virus cytotoxic drug.
2. application as claimed in claim 1; It is characterized in that: described anti-hepatitis C medicine be with the arteannuin or derivatives thereof as active constituents of medicine, process tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, nanometer formulation, any pharmaceutically acceptable dosage form of injection.
3. application as claimed in claim 1 is characterized in that: the derivant of described arteannuin comprises dihydroarremisine, Artemether, and artesunate, arteether is in the derivant of interior arteannuin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104434803A (en) * | 2013-09-13 | 2015-03-25 | 重庆汇智药物研究院有限公司 | Artesunate and L-lysine composition for injection and preparation method therof |
| CN104784167A (en) * | 2015-04-02 | 2015-07-22 | 河南黑马动物药业有限公司 | Medicine for treating animal eperythrozoonosis and preparation method thereof |
| CN114246858A (en) * | 2020-09-21 | 2022-03-29 | 北京化工大学 | Application of artemisinin compound in treatment and prevention of coronavirus infection |
| WO2022151551A1 (en) * | 2021-01-13 | 2022-07-21 | 上海交通大学 | Use of compound using intra-cyclic peroxo-bridged sesquiterpenes as parent nucleus in metabolism-related fatty liver disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1711091A (en) * | 2002-10-31 | 2005-12-21 | 凯敏食品公司 | The use of endoperoxides for the treatment of infections caused by flaviviridae, including hepatitis c, bovine viral diarrhea and classical swine fever virus |
| CN1758905A (en) * | 2003-02-12 | 2006-04-12 | 乔治敦大学 | Use of artemisinin for treating tumors induced by oncogenic viruses and for treating viral infections |
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2012
- 2012-07-16 CN CN2012102433696A patent/CN102755316A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1711091A (en) * | 2002-10-31 | 2005-12-21 | 凯敏食品公司 | The use of endoperoxides for the treatment of infections caused by flaviviridae, including hepatitis c, bovine viral diarrhea and classical swine fever virus |
| CN1758905A (en) * | 2003-02-12 | 2006-04-12 | 乔治敦大学 | Use of artemisinin for treating tumors induced by oncogenic viruses and for treating viral infections |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104434803A (en) * | 2013-09-13 | 2015-03-25 | 重庆汇智药物研究院有限公司 | Artesunate and L-lysine composition for injection and preparation method therof |
| CN104784167A (en) * | 2015-04-02 | 2015-07-22 | 河南黑马动物药业有限公司 | Medicine for treating animal eperythrozoonosis and preparation method thereof |
| CN114246858A (en) * | 2020-09-21 | 2022-03-29 | 北京化工大学 | Application of artemisinin compound in treatment and prevention of coronavirus infection |
| WO2022151551A1 (en) * | 2021-01-13 | 2022-07-21 | 上海交通大学 | Use of compound using intra-cyclic peroxo-bridged sesquiterpenes as parent nucleus in metabolism-related fatty liver disease |
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Application publication date: 20121031 |