WO2012058795A1 - 不饱和脂肪酸用于抑制病毒复制和/或感染的用途 - Google Patents

不饱和脂肪酸用于抑制病毒复制和/或感染的用途 Download PDF

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WO2012058795A1
WO2012058795A1 PCT/CN2010/078317 CN2010078317W WO2012058795A1 WO 2012058795 A1 WO2012058795 A1 WO 2012058795A1 CN 2010078317 W CN2010078317 W CN 2010078317W WO 2012058795 A1 WO2012058795 A1 WO 2012058795A1
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fatty acid
virus
hepatitis
acid
unsaturated fatty
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PCT/CN2010/078317
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English (en)
French (fr)
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李楚源
林青
黄琳
王德勤
邓乔华
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广州白云山和记黄埔中药有限公司
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Priority to PCT/CN2010/078317 priority Critical patent/WO2012058795A1/zh
Priority to AU2010363574A priority patent/AU2010363574B2/en
Priority to CN201080069059.6A priority patent/CN103096885B/zh
Publication of WO2012058795A1 publication Critical patent/WO2012058795A1/zh

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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of unsaturated fatty acids for inhibiting viral replication or preventing and/or treating viral infections, and more particularly, the present invention relates to the use of unsaturated fatty acids for inhibiting replication or prevention of hepatitis viruses, particularly hepatitis C virus. / or use for the treatment of hepatitis virus (especially hepatitis C virus) infection.
  • Viral infection is a common disease that seriously affects people's health.
  • Viral hepatitis is caused by hepatitis C virus infection and is one of the most widespread infectious diseases in the world. According to its virus series, it is mainly divided into seven types: viral hepatitis of type ⁇ , B, C, D, E, H and G.
  • viral hepatitis the risk of hepatitis C is second only to hepatitis B in humans. Since the discovery of the hepatitis C virus in the medical community in 1989, 170 million people have been infected worldwide. The infection rate of hepatitis C in our country is 3.2%.
  • hepatitis C virus infection is also very harmful to humans.
  • drugs for treating hepatitis C there are mainly the following drugs for treating hepatitis C: (1) drugs that inhibit viral replication (anti-virus); (2) immunomodulators; (3) liver-protecting drugs. Immunomodulators and hepatoprotective drugs play a complementary role in the treatment of hepatitis C.
  • antiviral drugs interferon is an ideal drug for the treatment of hepatitis C, with an effective rate of 60%-70%.
  • interferon cannot be administered orally, and the cost of treatment is high, and causes serious side effects such as depression, verrucous dysfunction, induced autoimmune disease, interstitial pneumonia, eye diseases, and cardiovascular diseases. Its wide application.
  • Other antiviral drugs, such as glucomannan, acyclovir, etc. may be used. These drugs have uncertain clinical effects or side effects and are not widely used. Therefore, there is still an urgent need to develop new and effective anti-hepatitis C virus drugs.
  • Unsaturated fatty acids have multiple functions, including prevention of obesity (Paniagua JA, et al. Diabetes Care 2007, 30: 1717-23). Regulating lipoprotein metabolism (Zheng CY, et al. American Journal of Clinical Nutrition 2008, 88 (2): 272-81), anti-inflammatory (Song C, et al. Stress, 2004, 7(1): 43), anti-cardiovascular disease (Ter6s S, et al. Proc Natl Acad Sci USA 2008, 105 ( 37): 13811-13816; Bucher HC, et al. Am J Med, 2002, 112(4): 298), Antipsychiatry (Nemets B, et al.
  • Radix Isatidis is a commonly used traditional Chinese medicine that is known to be useful for the prevention and/or treatment of viral infections. For example, during the acute respiratory distress syndrome (SARS) in China and worldwide during the epidemic, Radix is widely used for the prevention of SARS. treatment. However, to date, no active ingredient in Radix isatidis has been identified.
  • SARS acute respiratory distress syndrome
  • Radix isatidis can exert its antiviral action at least through unsaturated fatty acids; specifically, the unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids; more specifically, The monounsaturated fatty acids include erucic acid, and the polyunsaturated fatty acids include linoleic acid (ie, linolenic acid).
  • Another object of the present invention is to provide use of an unsaturated fatty acid or a pharmaceutically acceptable salt or ester thereof for the preparation of a medicament for preventing or treating a viral infection.
  • the unsaturated fatty acid is a monounsaturated fatty acid.
  • the monounsaturated fatty acid contains from 12 to 32 carbon atoms. In yet another embodiment the monounsaturated fatty acid is erucic acid:
  • the unsaturated fatty acid is a polyunsaturated fatty acid.
  • the polyunsaturated fatty acid contains from 12 to 32 carbon atoms, and in yet another embodiment, the polyunsaturated fatty acid is linoleic acid: Linoleic acid
  • the unsaturated fatty acid is a combination of a monounsaturated fatty acid and a polyunsaturated fatty acid.
  • the monounsaturated fatty acid and the polyunsaturated fatty acid may be administered simultaneously, sequentially or separately.
  • the virus is selected from the group consisting of a hepatitis virus, an enterovirus, a respiratory virus, an adenovirus, a human herpesvirus, a human papillomavirus, and the like.
  • the virus is a hepatitis virus.
  • the hepatitis virus is selected from the group consisting of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis C virus or hepatitis G virus.
  • the hepatitis virus is a hepatitis C virus.
  • Another object of the present invention is to provide a method of preventing or treating a viral infection which comprises administering to a subject in need thereof a prophylactically or therapeutically effective amount of an unsaturated fatty acid or a pharmaceutically acceptable salt or ester thereof.
  • the unsaturated fatty acid is a monounsaturated fatty acid.
  • the monounsaturated fatty acid contains from 12 to 32 carbon atoms. In yet another embodiment, the monounsaturated fatty acid is erucic acid:
  • the unsaturated fatty acid is a polyunsaturated fatty acid.
  • the polyunsaturated fatty acid contains from 12 to 32 carbon atoms, and in yet another embodiment, the polyunsaturated fatty acid is linoleic acid: Linoleic acid
  • the unsaturated fatty acid is a combination of a monounsaturated fatty acid and a polyunsaturated fatty acid.
  • the monounsaturated fatty acid and the polyunsaturated fatty acid may be administered simultaneously, sequentially or separately.
  • the virus is selected from the group consisting of a hepatitis virus, an enterovirus, a respiratory virus, an adenovirus, a human herpesvirus, a human papillomavirus, and the like.
  • the virus is a hepatitis virus.
  • the hepatitis virus is selected from the group consisting of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis C virus or hepatitis G virus.
  • the hepatitis virus is a hepatitis C virus.
  • Still another object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising an unsaturated fatty acid or a pharmaceutically acceptable salt or ester thereof.
  • the pharmaceutical composition comprises a prophylactically or therapeutically effective amount of an unsaturated fatty acid or a pharmaceutically acceptable salt or ester thereof, and optionally a pharmaceutically acceptable carrier.
  • the unsaturated fatty acid is a monounsaturated fatty acid.
  • the monounsaturated fatty acid contains from 12 to 32 carbon atoms. In yet another embodiment, the monounsaturated fatty acid is erucic acid:
  • the unsaturated fatty acid is a polyunsaturated fatty acid.
  • the polyunsaturated fatty acid contains from 12 to 32 carbon atoms, and in yet another embodiment, the polyunsaturated fatty acid is linoleic acid: Linoleic acid
  • the unsaturated fatty acid is a combination of a monounsaturated fatty acid and a polyunsaturated fatty acid.
  • the monounsaturated fatty acid and the polyunsaturated fatty acid may be administered simultaneously, sequentially or separately.
  • the "unsaturated fatty acid” refers to a fatty acid comprising at least one carbon-carbon double bond. It should be understood that the unsaturated fatty acids include diastereomers, enantiomers, tautomers, and geometric isomers, and the geometric isomers are represented as "E” or "Z”. Configuration isomer or a mixture of E and Z isomers. Unsaturated fatty acids are classified into monounsaturated fatty acids and polyunsaturated fatty acids depending on the number of double bonds. Monounsaturated fatty acid (MUFA) refers to a fatty acid containing a carbon-carbon double bond. Polyunsaturated fatty acid (PUFA) refers to a fatty acid containing two or more carbon-carbon double bonds.
  • MUFA monounsaturated fatty acid
  • PUFA Polyunsaturated fatty acid
  • the unsaturated fatty acid may be a monounsaturated fatty acid.
  • the monounsaturated fatty acid may be a monounsaturated fatty acid having from 12 to 32 carbon atoms, preferably from 18 to 22 carbon atoms, more preferably from 18 or 22 carbon atoms, most preferably erucic acid (ie Cis-13-twenty two
  • Erucic acid can be prepared by any method known in the art, for example, from radix isatidis, vegetable oil, broccoli oil, mustard oil, or commercially available as an extract or compound preparation.
  • the unsaturated fatty acid may also be a polyunsaturated fatty acid.
  • the polyunsaturated fatty acid may be a polyunsaturated fatty acid having from 12 to 32 carbon atoms, preferably from 18 to 22 carbon atoms, more preferably from 18 or 22 carbon atoms, most preferably linoleic acid (ie 9, 12, 15-octadecatrienoic acid, or linolenic acid): Linoleic acid
  • the linoleic acid can be prepared by any method known in the art, for example, from plant or plant extracts (e.g., in radix isatidis or linseed oil), or can also be purchased from a supplier (e.g., Sigma-Aldrich).
  • the unsaturated fatty acid may be a combination of a monounsaturated fatty acid and a polyunsaturated fatty acid.
  • the monounsaturated fatty acid and the polyunsaturated fatty acid may be administered simultaneously, sequentially or separately. Wherein for such simultaneous administration, it is especially advantageous to formulate the pharmaceutical composition in a single dosage form for ease of administration and uniformity of dosage.
  • the single dosage form refers to a physically separate unit suitable as a single dose, each unit containing a predetermined amount of active ingredient in association with the ⁇ RTIgt; ⁇ / RTI> desired pharmaceutical carrier to produce the desired therapeutic effect. Examples of such unit dosage forms are tablets, capsules, pills, powders, suppositories, injection solutions or suspensions and the like.
  • the virus may be selected from the group consisting of hepatitis virus, enterovirus, respiratory virus, adenovirus, human blister virus, human papillomavirus.
  • the hepatitis virus may be selected from the group consisting of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis C virus or hepatitis G virus, preferably type C. Hepatitis virus.
  • the subject comprises a mammal, in particular a human.
  • a "pharmaceutically acceptable salt of the compound” is a salt which is suitable for contact with human or animal tissues without undue toxicity or carcinogenicity, preferably without irritation, allergic reaction or other problems or complications.
  • Pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts can be prepared by reaction with inorganic or organic acids. Examples of suitable inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid, and the like. Examples of the organic acid include capric acid, acetic acid, propionic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, syringic acid, etc.
  • Suitable pharmaceutically acceptable base addition salts include Metal salts prepared from lithium, sodium, potassium, magnesium, calcium, aluminum and zinc, etc. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, PA 1995.
  • the compounds of the invention are administered in a prophylactically or therapeutically effective amount.
  • a prophylactically or therapeutically effective amount refers to an amount necessary to at least partially achieve the desired effect, or delay the onset of the particular condition being treated, inhibit its progression, or completely terminate its onset or progression.
  • the dosage administered can vary depending on the route of administration, the age of the patient, the weight, the type and severity of the condition being treated, and the like, which are well known to those skilled in the art and can be determined only by routine experimentation.
  • the pharmaceutical composition of the present invention can be produced by any known method.
  • the components can be simply mixed by a conventional method.
  • a pharmaceutically acceptable carrier can optionally be added.
  • the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventionally used in the pharmaceutical field, such as excipients, fillers, binders, disintegrants, lubricants, antioxidants, coating agents, colorants, fragrances, surfaces. Active agent, etc.
  • the pharmaceutical composition of the present invention can be used in accordance with a conventional method of various preparation types.
  • the pharmaceutical composition may be formulated into any suitable form including, but not limited to, tablets, capsules, pills, powders, suspensions, granules, sprays, aerosols, pills, oral solutions. , injection, tea.
  • the pharmaceutical composition can be administered by any suitable route including, but not limited to, for oral, injection, rectal, parenteral, subcutaneous, intravenous, intramuscular, and the like.
  • the present invention will be further described below by taking erucic acid and linoleic acid as examples.
  • HCV replicon cell model This cell model is a clone obtained by transfecting a human hepatoma cell line Huh-7 with a synthetic HCV subgenomic replicon and culturing it with a medium containing G418, which can stably express a replicon.
  • RNA reference [1].
  • the cell strain used in this study was supplied by ATCC, USA, and the cells were cultured in DMEM containing 10% fetal bovine serum.
  • PCR primers are provided by ATCC Corporation of the United States. 1. 4 BIO-RAD Benchmark, BIO-RAD, Hoefer, SORVALL pico.
  • a normal cell control was used as a negative control, and a recombinant human interferon control (rIFNa-2b, concentration of 2 IU/ml) was used as a positive drug control, and 7 duplicate wells were set for each concentration.
  • the cells were collected 72 hours after drug administration, and the cell survival rate was determined by MTT assay.
  • the calculation method of inhibition rate (IC): IC 1 - (A drug treatment group / A negative control group) X 100% (see Table 1 for the results).
  • HCV-(lb) replicon cDNA plasmid pNNeo/3-5BRG was linearized by XBal I, extracted with phenol/chloroform, and used with Mega in
  • Vitro T7 TranscriptKit was transcribed into HCV replicon RNA in vitro, extracted by phenol/chloroform, recovered by ethanol precipitation, transfected into Huh7 cells with lipofectin, and screened by G418 (500 g/ml DMEM). After 2 to 3 weeks, G418-resistant cell clones are formed and developed into cell lines. Since the HCV replicon RNA structure is recombined with the Neo gene, it can express an anti-G418 enzyme, so cells containing HCV replicons can Growing in medium containing G418.
  • Table 2 Inhibition of Hepatitis C Virus Replicon by Erucic Acid and Linoleic Acid IC50, IC90, EC50, EC90, SI50

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Description

不饱和脂肪酸用于抑制病毒复制和 /或感染的用途
技术领域
本发明涉及不饱和脂肪酸用于抑制病毒复制或者预防和 /或治疗病毒感染 的用途， 更具体而言， 本发明涉及不饱和脂肪酸用于抑制肝炎病毒（特别是 丙型肝炎病毒）复制或者预防和 /或治疗肝炎病毒（特别是丙型肝炎病毒）感 染的用途。
背景技术
病毒感染是一种常见的疾病， 严重影响人们的健康。 病毒性肝炎是由肝 炎病毒感染所引起的， 是世界上流传广泛， 危害很大的传染病之一。 按照其 病毒系列主要分为曱型、 乙型、 丙型、 丁型、 戊型、 己型和庚型病毒性肝炎 7 种。 在病毒性肝炎中， 丙型病毒性肝炎对人类的危险仅次于乙型病毒性肝炎。 自 1989年医学界发现丙型肝炎病毒以来， 全球已经有 1.7亿人受到感染， 我 国一^ 群中丙型肝炎的感染率为 3.2%。 据临床研究发现， 急性丙型肝炎感 染后约 50%-70%可转为慢性， 20%的人最终发展为肝硬化或肝癌， 且其演变 为肝癌的速度较乙肝病毒来得快。 因此， 丙型肝炎病毒感染对人的危害性也 很大。
目前， 治疗丙型肝炎的药物主要有以下几种： （1 )抑制病毒复制 （抗病 毒） 的药物； （2 )免疫调节剂； （3 )保肝药物。 免疫调节剂和保肝药物起到 辅助治疗作用， 但不能从根本上治疗丙型肝炎。 在抗病毒药物中， 干扰素是 治疗丙型肝炎的较理想药物， 有效率可达 60%-70%。但是干扰素不能口服给 药， 治疗费用较高， 并且引起严重的副作用， 比如抑郁症、 曱状腺机能障碍、 诱发自身免疫病、 间质性肺炎、 眼疾患和心血管疾病等， 这大大限制了其广 泛应用。 也可使用其它抗病毒药物， 比如阿糖腺香、 阿昔洛韦等， 这些药物 的临床疗效不确定或副作用大， 还不能广泛应用。 因此， 仍然迫切需要开发 新的有效的抗丙型肝炎病毒的药物。
不饱和脂肪酸具有多种功能， 包括预防肥胖 （Paniagua JA， et al. Diabetes Care 2007, 30: 1717-23 ). 调节脂蛋白的代谢作用 （ Zheng CY， et al. American Journal of Clinical Nutrition 2008, 88(2) :272-81 )、 抗炎（ Song C， et al. Stress, 2004， 7(1):43 )、 抗心血管疾病（Ter6s S， et al. Proc Natl Acad Sci U S A 2008, 105(37):13811-13816; Bucher HC, et al. Am J Med, 2002, 112(4): 298 )、 抗精神病（Nemets B, et al. Am J Psychiatry, 2002, 159(3): 477; Per M, et al. SchizophrRes, 2001 , 49(3): 243 ) 、 抗糖尿病 ( Minami A, et al. Br J Nutr, 2002, 87:157 )和抗癌 ( Valeria Pala, et al. Journal of the National Cancer Institute 2001， 93(14):1088-1095; Novak TE， et al. Am J Physiol, 2003, 284:L84 )等作用。 但是， 对不饱和脂肪酸的抗 病毒作用的报道甚少。
板蓝根是一种常用中药， 已知其能够用于预防和 /或治疗病毒感染， 例如 在 2003年急性呼吸窘迫综合症（SARS )在中国和全世界流行期间， 板蓝根 被大量用于 SARS的预防和治疗。 然而迄今为止尚未鉴定出板蓝根中起抗病 毒作用的活性成分。 经过大量研究， 发明人意外地发现， 板蓝根至少可以通 过不饱和脂肪酸来发挥其抗病毒作用； 具体来说， 所述不饱和脂肪酸包括单 不饱和脂肪酸和多不饱和脂肪酸； 更具体来说， 所述单不饱和脂肪酸包括芥 酸， 所述多不饱和脂肪酸包括亚油烯酸（即亚麻酸）。
发明内容
本发明的一个目的在于提供不饱和脂肪酸或其可药用盐或酯用于抑 制病毒复制的用途。
本发明的另一个目的在于提供不饱和脂肪酸或其可药用盐或酯在制 备用于预防或治疗病毒感染的药物中的用途。
在一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸。
在另一个实施方案中， 所述单不饱和脂肪酸含有 12至 32个碳原子。 在又一个实施方 所述单不饱和脂肪酸为芥酸：
Figure imgf000003_0001
在一个实施方案中， 所述不饱和脂肪酸为多不饱和脂肪酸。
在另一个实施方案中， 所述多不饱和脂肪酸含有 12至 32个碳原子, 在又一个实施方案中， 所述多不饱和脂肪酸为亚油烯酸：
Figure imgf000004_0001
亚油烯酸
在又一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸与多不饱 和脂肪酸的组合。 所述单不饱和脂肪酸与多不饱和脂肪酸可同时、 依次或 分开施用。
在一个实施方案中， 所述病毒选自肝炎病毒、肠道病毒、呼吸道病毒、 腺病毒、 人类疱疹病毒、 人乳头瘤病毒等。
在一个优选实施方案中， 所述病毒为肝炎病毒。
在一个进一步优选的实施方案中， 所述肝炎病毒选自曱型肝炎病毒、 乙型肝炎病毒、 丙型肝炎病毒、 丁型肝炎病毒、 戊型肝炎病毒、 己型肝炎 病毒或庚型肝炎病毒。
在一个更优选的实施方案中， 所述肝炎病毒为丙型肝炎病毒。
本发明的另一个目的在于提供一种预防或治疗病毒感染的方法， 其包 括向有此需求的对象施用预防或治疗有效量的不饱和脂肪酸或其可药用 盐或酯。
在一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸。
在另一个实施方案中， 所述单不饱和脂肪酸含有 12至 32个碳原子。 在又一个实施方案中， 所述单不饱和脂肪酸为芥酸：
Figure imgf000004_0002
在一个实施方案中， 所述不饱和脂肪酸为多不饱和脂肪酸。
在另一个实施方案中， 所述多不饱和脂肪酸含有 12至 32个碳原子, 在又一个实施方案中， 所述多不饱和脂肪酸为亚油烯酸：
Figure imgf000005_0001
亚油烯酸
在又一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸与多不饱 和脂肪酸的组合。 所述单不饱和脂肪酸与多不饱和脂肪酸可同时、 依次或 分开施用。
在一个实施方案中， 所述病毒选自肝炎病毒、肠道病毒、呼吸道病毒、 腺病毒、 人类疱疹病毒、 人乳头瘤病毒等。
在一个优选的实施方案中， 所述病毒为肝炎病毒。
在一个进一步优选的实施方案中， 所述肝炎病毒选自曱型肝炎病毒、 乙型肝炎病毒、 丙型肝炎病毒、 丁型肝炎病毒、 戊型肝炎病毒、 己型肝炎 病毒或庚型肝炎病毒。
在一个更优选的实施方案中， 所述肝炎病毒为丙型肝炎病毒。
本发明的又一个目的在于提供包含不饱和脂肪酸或其可药用盐或酯 的药物组合物。 所述药物组合物包含预防或治疗有效量的不饱和脂肪酸或 其可药用盐或酯以及任选地可药用载体。
在一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸。
在另一个实施方案中， 所述单不饱和脂肪酸含有 12至 32个碳原子。 在又一个实施方案中， 所述单不饱和脂肪酸为芥酸：
Figure imgf000005_0002
在一个实施方案中， 所述不饱和脂肪酸为多不饱和脂肪酸。
在另一个实施方案中， 所述多不饱和脂肪酸含有 12至 32个碳原子, 在又一个实施方案中， 所述多不饱和脂肪酸为亚油烯酸：
Figure imgf000006_0001
亚油烯酸
在又一个实施方案中， 所述不饱和脂肪酸为单不饱和脂肪酸与多不饱 和脂肪酸的组合。 所述单不饱和脂肪酸与多不饱和脂肪酸可同时、 依次或 分开施用。
具体实施方式
根据本发明， 所述 "不饱和脂肪酸" 指包含至少一个碳碳双键的脂肪 酸。 应当理解的是， 所述不饱和脂肪酸包括非对映异构体、 对映异构体、 互 变异构体和几何异构体， 所述几何异构体表示为 "E" 或 "Z" 构型异构体或 E和 Z异构体混合物。 根据双键个数的不同， 不饱和脂肪酸分为单不饱和脂 肪酸和多不饱和脂肪酸。 单不饱和脂肪酸 ( monounsaturated fatty acid , MUFA )指含有一个碳碳双键的脂肪酸， 多不饱和脂肪酸（polyunsaturated fatty acid, PUFA )指含有两个或两个以上碳碳双键的脂肪酸。
根据本发明， 所述不饱和脂肪酸可以是单不饱和脂肪酸。
根据本发明， 所述单不饱和脂肪酸可以是含有 12至 32个碳原子、 优 选含有 18至 22个碳原子、 更优选含有 18或 22个碳原子的单不饱和脂肪 酸， 最优选芥酸（即顺式 -13-二十二
Figure imgf000006_0002
可通过本领域公知的任何方法制备芥酸， 例如从板蓝根、 菜油、 海甘 蓝油、 芥子油中提取， 也可从提取物或化合物制备商处商购。
根据本发明， 所述不饱和脂肪酸也可以是多不饱和脂肪酸。
根据本发明， 所述多不饱和脂肪酸可以是含有 12至 32个碳原子、 优 选含有 18至 22个碳原子、 更优选含有 18或 22个碳原子的多不饱和脂肪 酸， 最优选亚油烯酸（即 9， 12， 15-十八碳三烯酸， 或称为亚麻酸）：
Figure imgf000007_0001
亚油烯酸
可通过本领域公知的任何方法制备亚油烯酸， 例如从植物或植物提取 物 （如板蓝根或亚麻籽油中 ） 提取， 或者， 也可从供应商 （例如 Sigma-Aldrich ) 处购得。
根据本发明， 所述不饱和脂肪酸可以为单不饱和脂肪酸与多不饱和脂 肪酸的组合。 所述单不饱和脂肪酸与多不饱和脂肪酸可同时、 依次或分开 施用。 其中对于所述同时施用而言， 以方便施用和剂量均一的单一剂型配 制所述药物组合物是特别有利的。 所述单一剂型指适于作为单一剂量的物 理上分开的单位， 每个单位含有预定量的活性成分， 以与任选所需的药物 载体一起产生所期望的疗效。这样的单位剂型的实例是片剂、胶嚢、丸剂、 粉末、 栓剂、 注射溶液或混悬液等。
根据本发明， 所述病毒可选自肝炎病毒、 肠道病毒、 呼吸道病毒、 腺 病毒、 人类疱療病毒、 人乳头瘤病毒。
根据本发明， 所述肝炎病毒可选自曱型肝炎病毒、 乙型肝炎病毒、 丙 型肝炎病毒、 丁型肝炎病毒、 戊型肝炎病毒、 己型肝炎病毒或庚型肝炎病 毒， 优选为丙型肝炎病毒。
才艮据本发明， 所述对象包括哺乳动物， 尤其是人。
所述化合物的 "可药用盐，，是这样的盐， 其适用于接触人或动物的组织而 没有过度的毒性或致癌性， 优选没有刺激、 过敏反应或其它问题或并发症。 这些盐包括可药用的酸加成盐和碱加成盐。 合适的可药用酸加成盐可通过与 无机酸或有机酸反应来制备。合适的无机酸的实例包括盐酸、硫酸和磷酸等。 合适的有机酸的实例包括曱酸、 乙酸、 丙酸、 琥珀酸、 乳酸、 苹果酸、 酒石 酸、 柠檬酸、 富马酸、 马来酸、 丁香酸等。 合适的可药用碱加成盐包括由锂、 钠、 钾、 镁、 钙、 铝和锌制备的金属盐等。 可药用盐的其它信息可见于 Remington's Pharmaceutical Sciences , 第 19版， Mack Publishing Co., Easton, PA 1995。 本发明的化合物以预防或治疗有效量施用。 预防或治疗有效量指至少部 分实现所期望作用、 或延緩所治疗具体病症的发作、 抑制其发展、 或者完全 终止其发作或发展所必需的量。 施用剂量可根据用药途径、 患者年龄、 体 重、所治疗疾病的类型和严重程度等变化，这些因素是本领域技术人员众所 周知的， 并且仅通过常规实验就可确定。
本发明的药物组合物可通过任何已知的方法制备。 各组分可以用常规 方法简单混合。 根据本发明， 可任选地添加可药用载体。 所述药学上可接 受的载体是指药学领域常规的药物载体， 如赋形剂、 填充剂、 粘合剂、 崩 解剂、 润滑剂、 抗氧化剂、 包衣剂、 着色剂、 芳香剂、 表面活性剂等。
本发明的药物组合物可按照各种制剂类型的常规方法使用。 根据本发 明， 所述药物组合物可以配制成任何合适的形式， 包括但不限于片剂、 胶嚢 剂、 丸剂、 粉末、 混悬剂、 颗粒、 喷雾剂、 气雾剂、 滴丸剂、 口服液、 注 射剂、 茶剂。 根据本发明， 所述药物组合物可以任意合适的途径施用， 包 括但不限于用于口服、 注射、 经直肠、 胃肠外、 皮下、 静脉内、 肌肉内等。 为了更好地理解本发明， 下面将以芥酸和亚油烯酸为例对本发明作进 一步说明。
实施例
发明人已经发现， 亚油烯酸和芥酸是板蓝根的有效成分。 利用 HCV 复制子细胞模型，研究了芥酸和亚油烯酸对 HCV复制子的体外抑制作用。 材料与方法
1、 材料
1. 1 研究药物 亚油烯酸和芥酸， 购自 SIGMA公司。
1. 2 HCV复制子细胞模型 该细胞模型是用合成的 HCV亚基因组复制 子转染人肝癌细胞株 Huh-7 并通过用含 G418 的培养基培养而获得的克 隆， 这些克隆可持续表达复制子 RNA， 参考文献 [1]。 本研究使用的细胞 株由美国 ATCC公司提供， 细胞培养于含 10%胎牛血清的 DMEM中。
1. 3 主要试剂 荧光定量 PCR检测试剂盒 (TAKARA), 细胞计数试剂盒 CCK-8(Dojindo), SYBR Green I， Calibration(BIO-RAD) , PCR引物由 美国 ATCC公司提供。 1. 4 主要仪器 酶标仪（BIO-RAD Benchmark) , 荧光定量 PCR 仪 (BIO-RAD)， 电泳系统 (Hoefer)， 台式离心机 (SORVALL pico)。
2 实验方法
2. 1 细胞毒性实验 取长满的 Replicon细胞 1瓶， 加 0.25%胰蛋白酶消 化， 制备成单细胞悬液。 细胞计数， 并调整浓度为 l x lO5细胞 /mL， 接种 96孔板 (100pL/孔）。 细胞于 37V、 5%C02培养过夜， 次日弃去培养上清 液， 依次加入含有药物 （芥酸、 亚油烯酸） 的完全培养基， 药物最高浓度 为 20μΜ， 按 10倍进行稀释， 共 5个浓度， 每浓度 7孔。 同时设正常细胞 对照作为阴性对照， 重组人干扰素对照 （rIFNa-2b， 浓度为 2 IU/ml )作 为阳性药对照， 每种浓度设 7个复孔。 药物作用 72h后收集细胞， MTT 法测定细胞的存活率。 抑制率 (IC)的计算方法： IC=1-(A药物处理组 /A阴 性对照组） X 100% (结果见表 1 )。
2. 2 HCV 复制子细胞模型的建立： 含 HCV-(lb)复制子 cDNA 质粒 pNNeo/3-5BRG经 XBal I酶切线性化后， 经酚 /氯仿抽提后， 用 Mega in
Vitro T7 TranscriptKit进行体外转录成 HCV复制子 RNA，再经酚 /氯仿抽 提， 乙醇沉淀回收后， 用脂质体（ lipofectin)转染 Huh7细胞， 经 G418筛 选 (500 g/ml DMEM)， 约经 2 ~3周后， 有耐 G418的细胞克隆形成， 将 其发展成细胞株， 由于 HCV复制子 RNA结构中重组有 Neo基因， 因此 能表达抗 G418的酶，故含有 HCV复制子的细胞能够在含 G418的培养基 中生长。
2. 3 Trizol法提取 HCV RNA， 参考文献 [2]， 提取细胞 HCV RNA。 荧 光定量 PCR反应检测 HCV RNA拷贝数， 与对照组比较，计算抑制率 (IC) 和半数有效剂量 (EC50)。 IC=1- (给药组拷贝数 /阴性对照组拷贝数） X 100%; EC50使用 Logit法计算（结果见表 2 )。
2. 4 荧光定量 PCR反应检测 HCV RNA[2]„
参考文献
[1] 巨立中，成军，钟彦伟.丙型肝炎病毒复制模型系统 [J].世界华人消化杂 志， 2003，11(12):1954-1956.
[2] 王健，赵金红，江水清，等.慢性乙肝患者 CXC趋化因子 IP-10的表达 [J]. 中华微生物学和免疫学杂志，2006,26(12) :1049-1050.
3 结果
3. 1 药物的细胞毒性作用 芥酸、 亚油烯酸在最高浓度 20μΜ 时细胞毒 性分别为 100.8%、 107%, 而对照药物重组人干扰素 rIFNa-2b在浓度 2 IU/ml时， 细胞毒性为 95.3%。
3. 2 药物对 HCV的抑制作用 芥酸、 亚油烯酸对 HCV复制子 RNA具 有抑制作用。 芥酸 20μΜ可导致 74.6% HCV RNA减少， 亚油烯酸 20μΜ 可导致 93.7% HCV RNA减少，对照药物重组人干扰素 rIFNa-2b在浓度 2 IU/ml时可导致 95.3% HCV RNA减少。 结果见表 1。 芥酸、 亚油烯酸对 HCV RNA的抑制程度与芥酸、 亚油烯酸剂量相关， 见表 2。
芥酸和亚油烯酸对丙型肝炎病毒复制子的抑制作用 化合物 浓度 活性 细胞毒性
(抑制％) (细胞％ ) 芥酸 20μΜ 74.6 100.8 亚油烯酸 20μΜ 93.7 107 阳性对照：重组 2 IU/ml 97 95.3%
人干扰素
( rIFNa-2b )
表 2: 芥酸和亚油烯酸对丙型肝炎病毒复制子抑制作用的 IC50、 IC90、 EC50、 EC90、 SI50 高测
测定方 细胞 浓度
化合物 测试类型 试浓 IC50 IC90 EC50 EC90 SI50 法 类型 单位
度
HCV 复 Huh7 确证性
芥酸 20 μΜ >20 >20 0.45 >20 >44.400 制子 ET (剂量反应）
HCV复 Huh7 确证性
亚油烯酸 20 μΜ >20 >20 2.53 17.5 >7.910 制子 ET (剂量反应）
重组人干扰素 HCV 复 Huh7 确证性 IU/m
2 >2 >2 0.06 0.4 >34.300
( rIFNa-2b ) 制子 ET (剂量反应） L 由表 1和表 2的结果可见， 芥酸和亚油烯酸均具有显著的丙型肝炎病 毒复制抑制作用， 尤其是亚油烯酸， 有开发为药品的潜力。 这为预防或治 疗肝炎提供了参考资料。
然而， 本领域技术人员应当理解的是， 提供这些实施例的目的仅在于 举例说明， 它们不以任何方式限制本发明的范围。 凡是在本发明范围内所 做出的具体实施方式及应用上的各种变动， 都落在本发明的范围内。

Claims

权 利 要 求
1. 不饱和脂肪酸或其可药用盐或酯用于抑制病毒复制的用途。
2. 不饱和脂肪酸或其可药用盐或酯在制备用于预防或治疗病毒感染 的药物中的用途。
3. 权利要求 1或 2的用途， 其中所述不饱和脂肪酸为单不饱和脂肪 酸， 优选为含有 12至 32个碳原子的单不饱和脂肪酸， 更优选为芥酸； 所 述不饱和脂肪酸也可以为多不饱和脂肪酸， 优选为含有 12至 32个碳原子 的多不饱和脂肪酸， 更优选为亚油烯酸。
4. 权利要求 1或 2的用途， 其中所述不饱和脂肪酸为单不饱和脂肪 酸与多不饱和脂肪酸的组合， 所述单不饱和脂肪酸与多不饱和脂肪酸可同 时、 依次或分开施用。
5. 权利要求 1至 4中任一项的用途， 其中所述病毒选自肝炎病毒、 肠道病毒、 呼吸道病毒、 腺病毒、 人类疱疹病毒、 人乳头瘤病毒， 优选为 肝炎病毒， 所述肝炎病毒选自曱型肝炎病毒、 乙型肝炎病毒、 丙型肝炎病 毒、 丁型肝炎病毒、 戊型肝炎病毒、 己型肝炎病毒或庚型肝炎病毒， 优选 为丙型肝炎病毒。
6. 一种药物组合物， 其包含预防或治疗有效量的不饱和脂肪酸或其 可药用盐或酯以及任选地可药用载体。
7. 权利要求 6的药物组合物， 其中所述不饱和脂肪酸为单不饱和脂 肪酸， 优选为含有 12至 32个碳原子的单不饱和脂肪酸， 更优选为芥酸； 所述不饱和脂肪酸也可以为多不饱和脂肪酸， 优选为含有 12至 32个碳原 子的多不饱和脂肪酸， 更优选为亚油烯酸。
8. 权利要求 6的药物组合物， 其中所述不饱和脂肪酸为单不饱和脂 肪酸与多不饱和脂肪酸的组合， 所述单不饱和脂肪酸与多不饱和脂肪酸同 时、 依次或分开施用。
9. 权利要求 6至 8中任一项的药物组合物， 其中所述药物组合物是 片剂、 胶嚢剂、 丸剂、 粉末、 混悬剂、 颗粒、 喷雾剂、 气雾剂、 滴丸剂、 口服液、 注射剂、 茶剂或其它常用的剂型； 其中所述药物组合物通过口服、 注射、 经直肠、 胃肠外、 皮下、 静脉内、 肌肉内等施用途径施用。
10.权利要求 6至 8中任一项的药物组合物， 其中所述药物组合物用于 抑制病毒复制或者预防和 /或治疗病毒感染。
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