AU2010363574A1

AU2010363574A1 - Uses of unsaturated fatty acids for inhibiting virus replication and /or infection

Info

Publication number: AU2010363574A1
Application number: AU2010363574A
Authority: AU
Inventors: Qiaohua Deng; Lin Huang; Chuyuan Li; Qing Lin; Deqin Wang
Original assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Current assignee: Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Co Ltd
Priority date: 2010-11-02
Filing date: 2010-11-02
Publication date: 2013-05-02
Anticipated expiration: 2030-11-02
Also published as: CN103096885A; WO2012058795A1; AU2010363574B2; CN103096885B

Abstract

Uses of unsaturated fatty acid linoleic acid and docosenoic acid or pharmaceutically acceptable salts or esters thereof for inhibiting hepatitis C virus replication or preventing and/or treating hepatitis C virus inflection are disclosed.

Description

-1/17 Uses of unsaturated fatty acids for inhibiting virus replication and/or infection FIELD OF INVENTION The present invention relates to use of an unsaturated fatty acid for the suppression of virus replication or the prevention and/or treatment of virus infection. Particularly, the present invention relates to use of an unsaturated fatty acid for the suppression of hepatitis virus ( in particular, hepatitis C virus) replication or the prevention and/or treatment of hepatitis virus ( in particular, hepatitis C virus) infection. BACKGROUND OF THE INVENTION Virus infection is a common disease that has significantly threaten human health. Viral hepatitis is caused by the infection of hepatitis virus, and is one of the most widely-spreaded, harmful infectious diseases around the world. Base on the type of virus, viral hepatitis may be mainly divided into 7 types, including hepatitis A, B, C, D, E, F and G. Among all viral hepatitises, hepatitis C is the second most harmful hepatitis for human after hepatitis B. 170 million people have been infected by hepatitis C virus all over the world since it was discovered in 1989, and in China, the infection rate of hepatitis C is 3.2% in general population. Based on clinical research, about 50%-70% patients of acute hepatitis C may turn into chronic hepatitis C, and 20% patients will finally develop into hepatocirrhosis or liver cancer, and its carcinogenesis is faster than that of hepatitis B virus. As a result, the infection of hepatitis C virus is very harmful to human. At present, the medicaments for the treatment of hepatitis C generally include: (1) medicaments for the suppression of virus replication (antivirus medicines); (2) immunomodulators; (3) -2/17 medicaments for liver protection. The immunomodulators and the medicaments for liver protection may be used as adjuvant treatment in the treating of hepatitis C, but hepatitis C cannot be thoroughly cured. Among all antivirus medicaments, interferon is considered as an ideal medicament for the treatment of hepatitis C with an effective rate up to 60%-80%. However, interferon can not be administrated orally and has several disadvantages including high cost and severe side effects, e.g., depression, dysthyroidism, and induction of autoimmune disease, interstitial pneumonia, eye diseases and cardiovascular diseases etc., which greatly limit the extensive application of interferon. Other antivirus medicaments may also be employed, e.g., arabinosyl adenosine, Aciclovir etc., and the clinical effects of these medicaments are uncertain or they have severe side effects, which are thus not allowed for extensive application. Therefore, there is still a need for developing a novel medicament with favorable anti-hepatitis C virus effect. There are a plurality of functions for unsaturated fatty acids, including obesity prevention (Paniagua JA, et al. Diabetes Care 2007, 30: 1717-23), regulation of the metabolism of lipoproteins (Zheng CY, et al. American Journal of Clinical Nutrition 2008, 88(2):272-81), anti-inflammation (Song C, et al. Stress, 2004, 7(1):43), anti-cardiovascular diseases (Terds 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), anti-psychosis (Nemets B, et al. Am J Psychiatry, 2002, 159(3): 477; Per M, et al. SchizophrRes, 2001, 49(3): 243), anti-diabetes (Minami A, et al. Br J Nutr, 2002, 87:157) and anti-tumor (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), and the like. However, few reports about the antivirus effect of unsaturated fatty acids have been reported. It is known that, as a common Chinese traditional medicine, Radix Isatidis may be used for the prevention and/or treatment of virus infection, and for example, during the prevalence of severe acute respiratory syndrome (SARS) in China and all over the world in 2003, Radix Isatidis -3/17 is widely used for the prevention and treatment of SARS. So far, however, the active ingredient in Radix Isatidis has not yet been identified. After a great amount of studies, the present inventor has unexpectedly found that the antivirus effect of Radix Isatidis may be achieved at least by unsaturated fatty acids; and particularly, the unsaturated fatty acids include monounsaturated fatty acids and polyunsaturated fatty acids; and more particularly, the monounsaturated fatty acids include erucic acid, and the polyunsaturated fatty acids include linolenic acid. Summary One purpose of the present invention is to provide use of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof for the suppression of virus replication. Another purpose of the present invention is to provide use of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof in preparation of the medicaments for the prevention or treatment of virus infection. In one embodiment, said unsaturated fatty acid is a monounsaturated fatty acid. In another embodiment, said monounsaturated fatty acid contains 12 to 32 carbon atoms. In another embodiment, said monounsaturated fatty acid is erucic acid: H OH H LM Erucic acid In one embodiment, said unsaturated fatty acid is a polyunsaturated fatty acid. In another embodiment, said polyunsaturated fatty acid contains 12 to 32 carbon atoms.

-4/17 In another embodiment, said polyunsaturated fatty acid is linolenic acid: 0 H3C Linolenic acid In another embodiment, said unsaturated fatty acid is the combination of a monounsaturated fatty acid and a polyunsaturated fatty acid. Said monounsaturated fatty acid and said polyunsaturated fatty acid may be administrated simultaneously, sequentially or separately. In one embodiment, said virus is selected from the group consisting of hepatitis virus, enterovirus, respiratory tract virus, adenovirus, human herpes virus, human papillomavirus and the like. In one preferable embodiment, said virus is hepatitis virus. In one further preferable embodiment, said 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 F virus and hepatitis G virus. In one more preferred embodiment, said hepatitis virus is hepatitis C virus. Another purpose of the present invention is to provide a method for the prevention or treatment of virus infection, which comprises administration of a prophylactically or therapeutically effective amount of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof to the subject in need thereof In one embodiment, said unsaturated fatty acid is a monounsaturated fatty acid. In another embodiment, said monounsaturated fatty acid contains 12 to 32 carbon atoms. In another embodiment, said monounsaturated fatty acid is erucic -5/17 acid: H OH Erucic acid In one embodiment, said unsaturated fatty acid is a polyunsaturated fatty acid. In another embodiment, said polyunsaturated fatty acid contains 12 to 32 carbon atoms. In another embodiment, said polyunsaturated fatty acid is linolenic acid: 0 -- ' OH H3C " Linolenic acid In another embodiment, said unsaturated fatty acid is the combination of a monounsaturated fatty acid and a polyunsaturated fatty acid. Said monounsaturated fatty acid and said polyunsaturated fatty acid may be administrated simultaneously, sequentially or separately. In one embodiment, said virus is selected from the group consisting of hepatitis virus, enterovirus, respiratory tract virus, adenovirus, human herpes virus, human papillomavirus and the like. In one preferable embodiment, said virus is hepatitis virus. In one further preferable embodiment, said 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 F virus and hepatitis G virus. In one more preferred embodiment, said hepatitis virus is hepatitis C virus.

-6/17 Another purpose of the present invention is to provide a pharmaceutical composition comprising an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof. Said pharmaceutical composition comprises a prophylactically or therapeutically effective amount of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof and optionally a pharmaceutically acceptable carrier. In one embodiment, said unsaturated fatty acid is a monounsaturated fatty acid. In another embodiment, said monounsaturated fatty acid contains 12 to 32 carbon atoms. In another embodiment, said monounsaturated fatty acid is erucic acid: H - OH Erucic acid In one embodiment, said unsaturated fatty acid is a polyunsaturated fatty acid. In another embodiment, said polyunsaturated fatty acid contains 12 to 32 carbon atoms. In another embodiment, said polyunsaturated fatty acid is linolenic acid: OH Linolenic acid In another embodiment, said unsaturated fatty acid is the combination of a monounsaturated fatty acid and a polyunsaturated fatty acid. Said monounsaturated fatty acid and polyunsaturated fatty acid may -7/17 be administrated simultaneously, sequentially or separately. DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the present invention, the "unsaturated fatty acid" is intended to mean the fatty acid comprising at least one carbon-carbon double bond. It should be understood that said unsaturated fatty acid includes diastereoisomers, enantiomers, tautomers and geometrical isomers, in which said geometrical isomers are expressed as "E" or "Z" isomers, or the mixture of E and Z isomers. According to the number of double bond, unsaturated fatty acid may be divided into monounsaturated fatty acid and polyunsaturated fatty acid. Monounsaturated fatty acid (MUFA) refers to the fatty acid containing one carbon-carbon double bond, while polyunsaturated fatty acid (PUFA) refers to the fatty acid containing two or more carbon-carbon double bonds. According to the present invention, said unsaturated fatty acid may be monounsaturated fatty acid. According to the present invention, said monounsaturated fatty acid may be the monounsaturated fatty acid comprising 12 to 32 carbon atoms, preferably 18 to 22 carbon atoms, more preferably 18 or 22 carbon atoms, and most preferably, the monounsaturated fatty acid may be erucic acid (i.e., cis-13-docosenoic acid): H OHH H Erucic acid Erucic acid may be prepared by any methods known in the art, for example, extracted from Radix Isatidis, rapes oil, crambe oil and mustard oil, or purchased from the manufacturer for the extract or the compound. According to the present invention, said unsaturated fatty acid may also be polyunsaturated fatty acid.

-8/17 According to the present invention, said polyunsaturated fatty acid may be the polyunsaturated fatty acid comprising 12 to 32 carbon atoms, preferably 18 to 22 carbon atoms, more preferably 18 or 22 carbon atoms, and most preferably, the polyunsaturated fatty acid may be linolenic acid (i.e., 9,12,15-octadecatrienoic acid): 0 HsC Linolenic acid Linolenic acid may be prepared by any methods known in the art, for example, extracted from plant or plant extracts ( such as Radix Isatidis or flaxseed oil), or purchased from a supplier (e.g., Sigma-Aldrich). According to the present invention, said unsaturated fatty acid may be the combination of monounsaturated fatty acid and polyunsaturated fatty acid. Said monounsaturated fatty acid and said polyunsaturated fatty acid may be administrated simultaneously, sequentially or separately. Among others, for simultaneous administration, it is particularly favorable to prepare said pharmaceutical composition in a convenient, homogeneous single formulation. Said single formulation is intended to mean physically separate unit that is suitable for single dose, and in each unit a predetermined amount of active ingredient is contained so as to achieve expected treatment effect together with optionally an appropriate pharmaceutical carrier. Examples of such unit formulation are tablet, capsule, pill, powder, suppository, injection solution or suspension etc. According to the present invention, said virus may be selected from the group consisting of hepatitis virus, enterovirus, respiratory tract virus, adenovirus, human herpes virus and human papillomavirus. According to the present invention, said 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 F virus and -9/17 hepatitis G virus, and preferably, it may be hepatitis C virus. According to the present invention, said subject includes mammals, particularly, the human. "Pharmaceutically acceptable salts" of said compound are the salt suitable for contacting human or animal tissues without excessive toxicity or carcinogenicity, preferably without stimulation, anaphylaxis or any other problem or complication. Such salts include pharmaceutically acceptable acid-addition salt and alkali-addition salt. Suitable pharmaceutically acceptable acid-addition salt may be prepared by the reaction with inorganic acid or organic acid. Examples of the suitable inorganic acid include hydrochloric acid, sulphuric acid and phosphoric acid etc. Examples of suitable organic acid include formic acid, acetic acid, propanoic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, and syringic acid etc. Suitable pharmaceutically acceptable alkali-addition salts include the metal salts prepared from lithium, sodium, kalium, magnesium, calcium, aluminum and zinc etc. Other information on the pharmaceutically acceptable salt may be found in Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, PA 1995. The compound of the present invention is administrated in a prophylactically or therapeutically effective amount. A prophylactically or therapeutically effective amount is intended to mean the amount that is required for at least partially providing the expected effect, delaying the occurrence of the specific disorder treated, suppressing its progress, or completely stopping its occurrence or progress. The dosage may be varied based on the route of administration, the age and weight of the patient, the type and severity of the disease treated and the like, all of which are well-known to those skilled in the art and may be determined just by routine experiments. The pharmaceutical composition of the present invention may be prepared by any known methods. Each component may be simply mixed by a conventional method. According to the present invention, a -10/17 pharmaceutically acceptable carrier may be optionally added. Said pharmaceutically acceptable carrier is intended to mean a common pharmaceutical carrier in the art of pharmaceutics, such as excipient, filler, binder, disintegrant, lubricant antioxidant, coating agent, colorant, aromatic agent and surfactant etc. The pharmaceutical composition of the present invention may be applied according to the conventional usage method for each type of formulation. According to the present invention, said pharmaceutical composition may be formulated into any suitable forms, which include, but are not limited to, tablet, capsule, pill, powder, suspension, pellet, spraying agent, aerosol, dripping pill, oral solution, injection and medicinal tea. According to the present invention, said pharmaceutical composition may be administrated by any suitable routes, which include, but are not limited to, oral, injection, transrectal, parenteral, subcutaneous, intravenous, and intramuscular administration. For a better understanding, the present invention will be further described using erucic acid and linolenic acid as examples. Examples It has been found by the present inventor that both linolenic acid and erucic acid are the active components of Radix Isatidis. The in vitro inhibitory effect of erucic acid and linolenic acid on HCV replicon has been investigated using the cell model for HCV replicon. Materials and methods 1. Materials 1.1 The agents used for test Linolenic acid and erucic acid were purchased from SIGMA. 1.2 Cell model for HCV replicon This cell model was the clone obtained by transfecting human liver cancer cell strain Huh-7 using synthetic HCV sub-genome replicon and cultured in G418-containing medium, and these clones could express the -11/17 replicon RNA consistently [1]. The cell strain used in the present invention was supplied by ATCC (USA), and the cells were cultured in DMEM containing 10% fetal bovine serum. 1.3 Reagents PCR Fluorescence Quantitative detection kit (TAKARA), cell counting kit CCK-8 (Dojindo), SYBR Green I, Calibration (BIO-RAD), PCR primers supplied by ATCC Company (USA). 1.4 Instruments Microplate reader (BIO-RAD Benchmark), fluorescence quantitative PCR instrument (BIO-RAD), electrophoresis system (Hoefer), desktop centrifuge (SORVALL pico). 2. Experimental methods 2.1 Cytotoxicity experiment Single cell suspension was prepared by adding 0.25% trypsin to a flask for digestion in which Replicon cells were grown to full confluence. Cell counting was performed to the suspension, and then the concentration was adjusted to 1x105 cells/mL followed by inoculation to a 96-well plate (100tL/well). The cells were cultured overnight at 37'C in 5% C0 2 , and the supernatant of the culture was discarded in the following day. Complete medium, which contained the agent (that is, erucic acid, linolenic acid) at a series of 5 concentrations with a maximum of 20pM and diluted in 10-fold. Each concentration was tested in 7 wells. At the same time, normal cell control was used as the negative control, and recombinant human interferon control (rIFNa-2b, concentration of 2 IU/ml) was used as the positive control. Each concentration was repeated in 7 wells. Cells were collected at 72 h after administration of the agents, and the cell survival rate was determined by MTT. The suppression rate (IC) was calculated as follows: IC = 1 - (A medicament group/A negative control group) * 100% (the results are listed in Table 1). 2.2 Establishment of HCV replicon cell model -12/17 HCV-(lb) replicon containing cDNA plasmid pNNeo/3-5BRG was linearized by digestion with XBal I, and then was extracted by phenol/chloroform. The resulting product was in vitro transcribed into HCV replicon RNA using Mega In Vitro T7 TranscriptKit. After extraction by phenol/chloroform and recovery by ethanol precipitation, Huh7 cells were transfected by liposome (lipofectin). After about 2-3 weeks of screening by G418 (500tg/ml DMEM), G418-resistant cell clones were obtained. After grown to a cell strain, anti-G418 enzyme may be expressed due to the Neo gene in the recombinant HCV replicon RNA. As a result, the cells containing HCV replicon may be grown in the medium comprising G418. 2.3 Extraction of HCV RNA by Trizol method According to a reference document [2], HCV RNA of cells was extracted. The number of HCV RNA copies was detected by fluorescence quantitative PCR reaction, and the suppression rate (IC) and half maximal effective concentration (EC50) were calculated by comparing with the control group. IC = 1 - (the number of copies in the administration group/the number of copies in the negative control group) * 100%; EC50 was calculated using Logit method (the results were listed in table 2). 2.4 Detection of HCV RNA by fluorescence quantitative PCR reaction [2]. References [1] Ju Lizhong, Cheng Jun, Zhong Yanwei. System of hepatitis C virus replication model. World Chinese Journal of Digestology, 2003, 11(12):1954-1956. [2] Wang Jian, Zhao Jinhong, Jiang Shiuqing, et al., Expression of CXC chemokine IP-10 in patients with chronic hepatitis B. Chinese Journal of Microbiology and Immunology, 2006, 26(12):1049-1050. 3. Results 3.1 Cytotoxicity of the medicaments -13/17 At the maximum concentration of 20pM, the cytotoxicity of erucic acid and linolenic acid was 100.8% and 107%, respectively, whereas the cytotoxicity of the control agent (recombinant human interferon rIFNa-2b) was 95.3% at the concentration of 2 IU/ml. 3.2 Inhibitory effect of the agents on HCV Both erucic acid and linolenic acid have inhibitory effect on HCV replicon RNA. 74.6% and 93.7% reduction of HCV RNA were obtained by erucic acid and linolenic acid at 20tM, respectively, and at the concentration of 2 IU/ml, the control agent (recombinant human interferon rIFNa-2b) resulted in a 95.3% reduction of HCV RNA. The results are listed in table 1. The inhibitory effects of erucic acid and linolenic acid on HCV RNA were correlated to the dosages of erucic acid and linolenic acid, as seen in table 2. Table 1. Inhibitory effects of erucic acid and linolenic acid on hepatitis C virus replicon Activity cytotoxicity (suppression%) (cell%) Erucic acid 20pM 74.6 100.8 Linolenic acid 20pM 93.7 107 Positive control: recombinant human 2 IU/ml 97 95.3% interferon (rIFNa-2b) Table 2. The inhibitory effects of erucic acid and linolenic acid on hepatitis C virus replicon: IC50, IC90, EC50, EC90 and S150 Detectio Cell High test Unit of Compound Test types IC50 IC90 EC50 EC90 S150 n method types concentration concentration Confirmatory HCV >44.4 Erucic acid Huh7 ET (dosage 20 M >20 >20 0.45 >20 Replicon 00 response) HCV Confirmatory >7.91 Linolenic acid Huh7 ET 20 M >20 >20 2.53 17.5 Replicon (dosage 0 -14/17 response) Positive control: recombinant human HCV Cofraoy>34.3 Huh7 ET (dosage 2 IU/mL >2 >2 0.06 0.4 interferon Replicon 00 response) (rlFNa-2b) As may be seen in Table 1 and Table 2, both erucic acid and linolenic acid (especially linolenic acid) have significant inhibitory effects on hepatitis C virus replication. Thus, they have great potential to be developed as a medicine. These findings provide a reference for the prevention or treatment of hepatitis. However, it should be understood by the one of skill in the art that these Examples are only for the purpose of illustration, and do not limit the scope of the present invention in any way. Any modifications may be made to the specific embodiments and applications under the scope of the present invention, which would fall into the scope of the present invention.

Claims

1. Use of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof for suppression of virus replication.

2. Use of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof in preparation of the medicaments for the prevention or treatment of virus infection.

3. The use of claim 1 or 2, wherein said unsaturated fatty acid is a monounsaturated fatty acid, and preferably a monounsaturated fatty acid comprising 12 to 32 carbon atoms, and more preferably erucic acid; said unsaturated fatty acid may also be a polyunsaturated fatty acid, and preferably a polyunsaturated fatty acid comprising 12 to 32 carbon atoms, and more preferably linolenic acid.

4. The use of claim 1 or 2, wherein said unsaturated fatty acid is the combination of a monounsaturated fatty acid and a polyunsaturated fatty acid, and said monounsaturated fatty acid and polyunsaturated fatty acid may be administrated simultaneously, sequentially or separately.

5. The use of any one of claims 1 to 4, wherein said virus is selected from the group consisting of hepatitis virus, enterovirus, respiratory tract virus, adenovirus, human herpes virus and human papillomavirus, and preferably is hepatitis virus, and said 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 F virus and hepatitis G virus, and preferably is hepatitis C virus.

6. A pharmaceutical composition, comprising a prophylactically or therapeutically effective amount of an unsaturated fatty acid or pharmaceutically acceptable salt or ester thereof and optionally a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein said unsaturated fatty acid is a monounsaturated fatty acid, and preferably a monounsaturated fatty acid comprising 12 to 32 carbon atoms, and more preferably erucic acid; said unsaturated fatty acid may also be a polyunsaturated fatty acid, and preferably a polyunsaturated fatty acid -16/17 comprising 12 to 32 carbon atoms, and more preferably linolenic acid.

8. The pharmaceutical composition of claim 6, wherein said unsaturated fatty acid is the combination of a monounsaturated fatty acid and a polyunsaturated fatty acid, and said monounsaturated fatty acid and polyunsaturated fatty acid may be administrated simultaneously, sequentially or separately.

9. The pharmaceutical composition of any one of claims 6 to 8, wherein said pharmaceutical composition is the form of tablet, capsule, pill, powder, suspension, pellet, spraying agent, aerosol, dripping pill, oral solution, injection, medicinal tea or any other common formulations; and wherein said pharmaceutical composition may be administrated by the following routes: oral, injection, transrectal, parenteral, subcutaneous, intravenous, intramuscular administration and the like.

10. The pharmaceutical composition of any one of claims 6 to 8, wherein said pharmaceutical composition is used for the suppression of virus replication or the prevention and/or treatment of virus infection.