The use of sesquiterpenes and, in particular sesquiterpene lactone endoperoxides, such as artemisinin and analogs thereof, for the treatment of hepatitis C virus infections. Artemisinin, analogs of artemsisnin and some crude Artemisia extracts were tested in vitro against DNA-viruses, retro-viruses and Flavivirida, (an important family of human and animal RNA pathogens). These compounds were also screened for anti-tumor activity. Strong activity of artemisinin was noticed against the bovine viral diarrhea virus (BVDV). As pestiviruses, such as BVDV, share many similarities with hepatitis C virus (HCV), we can conclude that endoperoxides in general and artemisinin more specificly have efficacy as treatments for hepatitis C viral infections.
The application of endoperoxide in the medicine of the infection that the preparation treatment is caused by flaviviridae (comprising hepatitis C virus, bovine viral diarrhea virus and swine fever virus)
Background of invention
The present invention relates generally to the application of the application, particularly sesquiterpene lactones endoperoxide of sesquiterpene lactones class (sesquiterpene lactones) in the infection that treatment is caused by flaviviridae (Flaviviridae) in treatment hepatitis C infection, yellow fever, dengue fever, bovine viral diarrhoea and swine fever (classical swine fever).
Chronic hcv infection is an important public health problem that influences the whole world 1.8 hundred million people (total population 3%), comprises 4 million peoples of the U.S. here, and is a main cause that causes the liver chronic disease.Someone expects that the HCV infection of the U.S. will continue to increase, and will reach present 3 times to the infected in 2010.The infection of hepatitis C virus may cause serious, may be life-threatening chronic hepatopathy in some cases, comprises liver failure and hepatocarcinoma.
In the U.S., chronic hepatopathy is to cause being grown up the tenth dead reason, and causes for 8 every year near 25000 examples or near 1% death toll.Studies show that on the population-based: 40% chronic hepatopathy is that HCV is relevant, causes the annual approximately death of 8000-10000.The medical treatment of the acute and chronic hepatopathy that HCV is relevant and work estimated amount of damage every year will be above 600,000,000 dollars.And the hepatopathy in late period that HCV is relevant is the modal reason of adult orthotopic liver transplantation.Because most HCV the infected's age, the death toll that is caused by the chronic hepatopathy relevant with HCV may significantly increase in the period of the 10-20 in future between 30-49 year, at this time this group the infected will reach the age that the complication of chronic hepatopathy takes place usually.
The propagation of HCV mainly is a large amount of or repeated direct by skin and blood.In the U.S., propagating two kinds of relevant modal contacts with HCV is blood transfusion and shoot up.Therapy for hepatitis C is to change field fast in the clinical practice.The therapeutic alliance of interferon and ribavirin (a kind of nucleic acid analog) has been approved for the treatment first of chronic hepatitis C.The patient of the therapeutic alliance of using ribavirin and interferon be studies confirm that compare with the response rate 15-25% of independent application of interference extract for treating, its persistent response rate has remarkable increase, reaches 40-50%.Most of patients has interferon one property crossed cold like symptoms in early days in treatment, but these symptoms alleviate in continuing treatment.Side effect subsequently has fatigue, bone marrow depression and maincenter mentation (for example, indifferent, cognitive variation, irascible and depressed).Because serious adverse, the dosage of the patient's of 10-40% interferon must reduce, and the patient of 5-15% must be interrupted use.Ribavirin can cause hemolytic anemia, and also is problematic for the unborn anemia of patient, bone marrow depression or renal failure.These patients, should avoid therapeutic alliance, maybe should attempt to correct anemia.The hemolytic anemia that ribavirin causes also may be life-threatening to the patient that ischemic heart disease or cerebrovascular disease are arranged.Ribavirin be cause odd-shaped, female patient in therapeutic process, to avoid gestation.Other treatment comprises corticosteroid, ursodeoxycholic acid (ursodiol) and thymosin, is invalid.The high iron level of liver can reduce the drug effect of interferon.The use in conjunction of de-iron treatment (venotomy or chelating art) and interferon after deliberation, still, also still do not have conclusion ( Www.cdc.gov).So, medical domain press for find with development can be used for treating HCV infect, have simultaneously less or can reduce side effect and compare the new bioactive molecule that better curative effect is arranged with existing treatment.
Summary of the invention
Have been found that sesquiterpene lactones with following general formula:
Have antagonism by flaviviridae, comprise the activity of the infection that hepatitis C virus, bovine viral diarrhea virus and swine fever virus cause.The application has described some representative, at present preferred sesquiterpene lactones classes, and but, those skilled in the art obviously can understand, and other sesquiterpene lactones chemical compounds also are effective in the treatment of the infection that is caused by flaviviridae in the known technology.The pharmaceutically acceptable salt that also comprises this compounds.
The sesquiterpene lactones of preferentially selecting for use is sesquiterpene lactones endoperoxides artemisinin (artemisinin), dihydroarteannuin (dihydroartemisinin), artesunate (artensunate) and Artemether (artemether).
Description of drawings
Fig. 1 is the graphic extension to arteannuin, dihydroarteannuin, artesunate and Artemether chemical constitution.
The specific embodiment
Among the application, we will introduce the antiviral effect of sesquiterpene lactones class, sesquiterpene lactones endoperoxide particularly, for example arteannuin, dihydroarteannuin, artesunate and Artemether are to the specific flaviviridae antivirus action of hepatitis C virus for example.
Preferred sesquiterpenoid of the present invention comprises the chemical compound with following general formula:
Wherein: X 1And X 2Be selected from O, S, Se and NH; Y is selected from O, S, Se and NH; Z is selected from O, NH, S and Se, and Q is selected from CO, CHOH, CHOCH 3, CHOC 2H 5, CHOC 3H 7And CHOCOCCH 2CH 2COOH and drug acceptable salt thereof.
The particularly preferred sesquiterpenoid of the present invention comprises arteannuin, its X 1And X 2Be that O, Y are that O, Z are that O and Q are C=O; Dihydroarteannuin (same arteannuin, but Q is CHOH); Artemether (same arteannuin, but Q is CHOCH 3); Arteether (arteether) (same arteannuin, but Q is COC 2H 5); A kind of propyl group product (same arteannuin, but Q is CHOC 3H 7); And artesunate (artesunate) (same arteannuin, but Q is CHOCOCH 2CH 2COOH).Most preferred sesquiterpenoid of the present invention is a dihydroarteannuin.
Endoperoxide has a dioxy key (peroxo linkage), and (O-O), to be considered to that these products are had the malaria activity be very important to this structure.With-S-S-(disulfide bond), or-Se-Se-(two selenium keys), or-N-O-or-NH-NH-(hydrazine), and the various combination of above-mentioned key can produce new chemical compound, also may have activity.
Flaviviridae is (the Rice CM.1996.Flaviviridae:the viruses and their replication.In:Fields BN of important RNA viruses pathogen section of human and animal, Knipe DM, Howley PM, eds.Fields virology.Philadelphia:Lippincott-Raven Publishers.Pp 931-960).The genus of the flaviviridae of having found at present shows different aspect route of transmission, host range and pathogeny.Typical banzi virus member is yellow fever virus, dengue virus and Pestivirus (pestiviruses), for example bovine viral diarrhea virus (BVDV) and swine fever virus (CSFV).The member of the tool feature that this family is up-to-date is people's a common and proprietary pathogen, i.e. hepatitis C virus (HCV).Banzi virus is to have (+) the polar single strand RNA virus of adopted rna gene group is arranged.Other have (+) has the Viraceae of adopted RNA to comprise Picornaviridae (Picornaviridae), Togaviridae (Togaviridae), Caliciviridae (Caliciviridae) and coronaviridae (Coronaviridae).
The compounds of this invention can be used their original shape or their salt.In certain situation, these chemical compounds can form the stable nontoxic acid or the salt of alkali by enough alkalescence or acidity, and these chemical compounds are used with the form of salt and are fit to.The example of the acceptable salt of pharmacy is can accept the acylate that anionic organic acid forms with the physiology who forms, ascorbic acid acetate for example, benzoate, citrate, etoglutarate, glycerophosphate, malonate, mesylate, succinate and tartrate.Suitable inorganic acid salt also can be synthesized, and comprises heavy carbonate, carbonate, hydrochlorate, nitrate, and sulfate.
The present invention can contain the pharmaceutical composition of described chemical compound and suitable excipient and conventional administration, and said composition antagonism viral infection is useful.According to what treat is interior or outer viral infection, and chemical compound of the present invention and compositions can be through parenteral, part, intravaginal, oral or rectally.
To parenteral, active component or its saline solution can prepare by water, randomly mix with nontoxic surfactant.Also can be at glycerol, liquid macrogol, the mixture of triacetin and mentioned reagent and in oil preparation, preparing.
The spendable dosage of chemical compound can be by determining in its activity of external comparison.Extrapolation method to people's effective dose is known in the art.
This chemical compound can unit dose the conventional administration of form; For example the per unit dosage form comprises active component 0.1-2000mg, is preferably 100-1000mg, most preferably is 100-500mg.Required dose can disposable routinely administration or with suitable interval gradation administration, for example can divide 2,3,4 or more times administration every day.Each sub-doses can also further separately for example be divided into the administration form of some dispersive loose separations; For example repeatedly suck or repeatedly put ocular administration from medicine feeding machine.
To infecting in the body, compositions can be passed through oral or parenteral by the dosage level that free drug calculates, and is approximately 1-30mg/kg (weight of mammal), preferred 1-10mg/kg (weight of mammal).
Must depend on the needs of the individuality of being treated in the accurate scheme of the administration of the chemical compound of this announcement and compositions, the type of treatment also has attending doctor's judgement certainly.Chemical compound of the present invention can be used for having the animal of this treatment demand.Under the situation, be to be used for the people mostly, but domestic animal and house pet also can be expected especially and belonged to range of application of the present invention.
Material and method
Arteannuin is purchased in Aldrich.The plant crude extract of Absinthium (Artemisia absinthum) KE-4, Herba Artemisiae annuae (Artemisia annua) KE-5 and tarragon (Artemisia dracunculus) KE-6 is from U.S. Des Moines, and Kemin Industrial Co., Ltd obtains.In order to prepare KE-4 ,-5 and-6 samples, ground Artemisia absinihium L, sweetness and bitterness Chinese mugwort or the tarragon that 2g is done stirring at room 8 hours in the 200ml hexane, then, suspension filter through the G3 glass filter and low pressure under evaporate.Residual black filtering residue is used for biological screening.Artemether, dihydroarteannuin and artesunate are purchased the Pharma in Dafra.Above-claimed cpd has all carried out the screening of anti-different Causative viruss, for example human immunodeficiency virus (HIV), herpes simplex virus (HSV), chickenpox virus (VV), varicella zoster virus (VZV) and human cytomegalic inclusion disease virus (CMV).To all virus, except CMV, detected EC 50(to HIV in people's cem cell culture inductive cytopathogenicity, HSV and VV at human embryonic fibroblast E 6Inductive cytopathogenicity and VZV inductive plaque in human embryonic lung (HEL) cell culture forms the concentration that reaches the required active compound of 50% inhibition in the SM cell culture).For determination of antiviral activity, anti-CMV IC 50Expression, the human embryonic lung becomes fiber (HEL) cell to cultivate every hole 20PFU viral infection with 96 orifice plates.
After 2 hours, add the culture fluid that 0.1ml contains the gradient dilution of test compounds 37 ℃ of cultivations in the cell of infection.After cell Giemsa dyeing, counted plaque on the 7th day at microscopically.Minimum antiviral concentration reaches 50% required dosage with the plaque formation that suppresses virus induction and represents.
These chemical compounds have also been carried out the screening of anti-flavivirus.Because in fact there are not enough analyzed in vitro to be used for anti-HCV screening, so decision screening anti-bovine viral diarrhea virus (BVDV), because it and hepatitis C virus have a lot of similarity (Frolovl, McBride S and Rice CM.Cis-acting RNA elements required for replication of bovine.Viral diarrheavirus-hepatitis C virus 5 ' non-translated region chimeras.RNA 4,1418-1435 (1998)).Further the effect of the not homophyletic of the anti-BVDV of Artemether is screened.
These chemical compounds have also been carried out the detection of anti-tumor activity, by (Molt4/C8, propagation CEM/0) (detection) is carried out to murine leukemia cell (L1210/0), molluscum contagiosum adenocarcinoma cell (FM3A) and human T lymphocyte.Observed best antiviral activity to CMV and VZV is from KE-6.Next is the antiviral activity of arteannuin to HSV-2 and VV.
Result and discussion
In first screening (seeing Table 1), detected arteannuin and some artemisia crude extract (KE-4, KE-5 and KE-6) antiviral activity to HIV-1, HIV-2, HSV-1, HSV-2, VV, CMV and VZV.
Table 1: the The selection result of anti-HIV-1, HIV-2, HSV-1, HSV-2, VV, CMV and VZV
aThe 50% couple of HIV in human lymphocyte cem cell culture inductive cytopathogenicity, HSV and VV at human embryonic fibroblast E 6Inductive cytopathogenicity and CMV and VZV inductive plaque in human embryonic lung's hel cell culture forms and reaches 50% the required compounds effective concentration of inhibition in the SM cell culture.
Carried out second screening (table 2) in addition, so that detect the activity of anti-bovine viral diarrhea virus in Ren Bovis seu Bubali (MDBK) cell.
Table 2: the The selection result of anti-BVDV in the MDBK cell
Antiviral activity is assessed on the MDBK cell with the Pe515 strain of BVDV.The MTS method is all used in antiviral activity and Cytotoxic detection.
EC 50Be needed concentration when reducing the cytotoxic effect 50% that virus causes.MTC (minimum toxic concentration) is defined as and causes that cellular metabolism reduces>=20% concentration.
When handling the MDBK cell with Artemether, arteannuin, KE4 and KE5, (100 μ g/ml) still do not reach MTC at maximum concentration.Observe the activity that endoperoxides artemisinin, dihydroarteannuin, Artemether and artesunate have very strong anti-flavivirus BVDV in this test, have low cytotoxicity simultaneously.So these products can be used for the treatment of the infection that is caused by banzi virus.
Carried out the screening (table 3) of anti-different B VDV strain and anti-BDV (border disease virus) with Artemether
Table 3: the The selection result of anti-BVDV of Artemether and BDV
Artemether
|
100 μg/ml
|
20 μg/ml
|
4 μg/ml
|
800 ng/ml
|
160 ng/ml
|
32 ng/ml
|
6.4 ng/ml
|
1.28 ng/ml
|
0.512 ng/ml
|
0.256 ng/ml
|
Non-infection
|
Toxicity
|
|
|
|
|
|
|
|
|
|
BVDVII ncp
|
Toxicity
|
++
|
++
|
++
|
+++
|
+++
|
+++
|
+++
|
+++
|
+++
|
BVDVI cp
|
Toxicity
|
+
|
+
|
+
|
+
|
++
|
++
|
++
|
++
|
++
|
BVDVI ncp
|
Toxicity
|
+
|
+
|
+
|
++
|
+++
|
+++
|
+++
|
+++
|
+++
|
BDV
|
Toxicity
|
+
|
+
|
+
|
++
|
++
|
++
|
++
|
++
|
++
|
+++all cells all is male
+++/-50% cell is male
++/-10% cell is male
The BVDV bovine viral diarrhea virus
The BDV border disease virus
The strain of cp cytopathogenic effect
The non-cytopathogenic effect strain of ncp
Anti-especially BVDVIcp (the BVDV strain the most similar to HCV) can be clear that the effect of Artemether.The EC of this product 90=0.16 μ g/ml.The toxic concentration of finding in these experiments is near 100 μ g/ml.Also can observe the effect (EC of anti-BDV 90=0.8 μ g/ml).
Arteannuin and artemisia crude extract (KE-4, KE-5 and KE-6) have all been carried out the screening of antitumor cell strain L12 10/0, FM3A/0, Molt4/C8 and CEM/0, but do not find useful result.
Conclusion
We do not find any tangible antiviral activity in initial resisting DNA virus and retroviral screening.We clearly illustrate the strong antiviral activity of arteannuin, artesunate, Artemether and dihydroarteannuin in the screening of the anti-BVDV second time (RNA viruses).These products all are endoperoxides, compare with the artemisia crude extract that detected, and show obviously better anti-BVDV activity.Because BVDV and HCV have many similaritys, arteannuin, artesunate, Artemether, dihydroarteannuin and other possible peroxide may have strong and the antiviral properties of anti-HCV optionally.
Although the present invention has described the preferred specific embodiment by expectation, be understandable that the present invention is not limited only to this, because to its variation made from modify and also to belong in the appended claims in the defined full breadth of the present invention.
1, sesquiterpene or the acceptable salt of its pharmacy purposes in a kind of medicine that is used for the treatment of the infection that causes by flaviviridae of preparation, described sesquiterpene has following general formula:
Wherein:
Q is selected from CO, CHOH, CHOCH 3, CHOC 2H 5, and CHOCOCCH 2CH 2COOH.
2, the purposes of claim 1, wherein said infection is a hepatitis C.
3, the purposes of claim 1, wherein said infection are bovine viral diarrhoea or swine fever.
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