WO2009072779A1

WO2009072779A1 - Composition comprising sodium meta-arsenite for treatment of hepatitis c

Info

Publication number: WO2009072779A1
Application number: PCT/KR2008/007028
Authority: WO
Inventors: Sang Bong Lee; Jong Wook Kim; Hyung-Sup Shim
Original assignee: Sang Bong Lee; Jong Wook Kim; Hyung-Sup Shim
Priority date: 2007-12-04
Filing date: 2008-11-28
Publication date: 2009-06-11
Also published as: KR20090058423A; CN101820888A

Abstract

The present invention relates to a composition comprising sodium meta-arsenite for the treatment of hepatitis C. More specifically, the present invention relates to a novel composition comprising sodium meta-arsenite having antiviral activity against hepatitis C virus thus being useful for the treatment of hepatitis C.

Description

COMPOSITION COMPRISING SODIUM META- ARSENITE FOR TREATMENT OF HEPATITIS C

Technical Field

[1] The present invention relates to a composition comprising sodium meta-arsenite for the treatment of hepatitis C. Background Art

[2] The contagious viral hepatitis C is mostly caused by contamination in blood or body fluids with hepatitis C virus (HCV), a small enveloped RNA virus belonging to Hep- acivirus genus of Flaviviridae family. Once infected with HCV, it often leads to chronic hepatitis in more than 85% of the infected people and also the prognosis is not good so that it may even develop serious liver-related diseases such as liver cirrhosis and liver cancer, unlike in other known types of hepatitis(e.g., A, B, D, and E).

[3] According to the WHO report, about 170 million people are already infected with the hepatitis C in the world, and an annual increase of about 25,000 people is being added to the list each year. In Korea, the hepatitis C patients are known to be about 450,000. Further, the number of liver transplant recipients due to HCV-related liver diseases account for about 40% of the total liver transplant recipients.

[4] In Korea, with the introduction of HCV screening on donated blood in 1992, the blood contamination through blood donation has been much reduced. Further, in addition to the existing patients, the increase in the number of new patients of hepatitis C due to inappropriate drug administration, vertical transmission, false negative diagnosis at the time of screening donated blood, has been raised as a new social issue.

[5] Unlike hepatitis A and B, there has not been developed any practical vaccine for the prevention of viral hepatitis C and it is not likely that the vaccine will be developed and commercialized in the near future. One of the main reason lies in that the hepatitis C virus dodges attack by the immune system in the body it entered by rapidly changing its shape into various variants. Further, agents such as alpha-interferon and ribavirin are being commercialized but their prices are very high. Therefore, there is an urgent need for the development of effective therapeutic agents for the treatment of hepatitis C. Disclosure of Invention

Technical Problem

[6] The inventors of the present invention, while performing numerous researches in search of a therapeutic drug to be commercialized for the effective treatment of the virus with low toxicity and a reasonable price, finally discovered that sodium meta- arsenite has an antiviral activities against hepatitis C virus and completed the present invention.

[7] Therefore, the objective of the present invention is to provide a composition comprising sodium meta-arsenite as an active ingredient for the treatment of hepatitis

C virus.

Technical Solution

[8] The present invention relates to a composition comprising sodium meta-arsenite as an active ingredient for the treatment of hepatitis C. Brief Description of the Drawings

[9] The above and other features of the present invention will now be described in detail with reference to certain exemplary embodiments thereof illustrated the accompanying drawings which are given hereinbelow by way of illustration only, and thus are not limitative of the present invention, and wherein:

[10] Fig. 1 shows a curve for Huh-7 cell growth inhibition according to various SMA concentrations along the time passage (Fig. IA) and a curve for cell viability (%) compared to that of the control group according to time passage along various SMA concentrations (Fig. IB);

[11] Fig. 2 shows pictures of cells 48 hours after treatment with various SMA concentrations;

[12] Fig. 3 shows pictures of cell nuclei staining with DAPI 48 hours and 72 hours respectively, after treatment with various SMA concentrations [phenomenon of apoptotic fragmentation is observed at a concentration of 1 M or higher] ;

[13] Fig. 4 and Fig. 5 show the change in the amount of NS5A, which is a protein of hepatitis C virus, measured by immuno-blotting of SMA (Hsp 70 and -tubulin, being cell proteins, are used as control);

[14] Fig. 6 shows the result of NS5A positive cells measured by immunofluorescenece staining; and

[15] Fig. 7 shows the change in the amount of NS5A detected by immuno-blotting according to various SMA concentrations (Fig. 7A) and along the time passage (Fig.

7B).

Best Mode for Carrying Out the Invention

[16] The present invention is described further as set forth hereunder.

[17] The present invention relates to a novel composition comprising sodium meta- arsenite ( SMA hereinafter), which has antiviral activity against hepatitis C virus, as an active ingredient to be effective for the treatment of hepatitis C.

[18] The SMA of the present invention is a sodium salt of meta-arsenous acid containing

AsO₂" and can be manufactured by reacting arsenic trioxide with sodium hydroxide. It has been shown to have an inhibitory activity against cancer cell growth.

[19] According to the test results of the present invention, with regard to the cytotoxicity of SMA , there was almost no cytotoxicity observed at the concentration of 0.5 M or less; about 40% of inhibition against cell growth was observed at the concentration of 1 M; and cell growth was completely terminated at the concentration of 5 M or more due to extremely high cytotoxicity.

[20] Further, the LD₅₀ the concentration of SMA when the cells treated with SMA for 3 days show 50% reduction in cell growth as compared to those not treated with SMA , was found to be about 3.5 M.

[21] Regarding the inhibitory activity against growth of hepatitis C virus, the treatment with SMA at the concentration of 0.5 - 1 M for 24 hours showed about 20 - 40% of antiviral activity; at the concentration of 2.5 M for 24 hours showed about 45 - 50% of antiviral activity; at the concentration of 5 - 10 M for 24 hours showed about 50 - 70% of antiviral activity; and the half maximal inhibitory concentration of SMA was found to be about 2.0 M.

[22] From the above, it was confirmed that SMA has an excellent anti-HCV activity thus being useful as an active ingredient for a composition for the treatment of hepatitis C.

[23] Therefore, the present invention relates to a composition comprising SMA for the treatment of hepatitis C.

[24] In manufacturing a pharmaceutical drug comprising SMA as an active ingredient, can further comprise other pharmaceutically acceptable carriers or excipients and be prepared in the form of tablets, granules, capsules, suspensions, emulsions, or can be formulated into drugs for unit parenteral administration or for parenteral administration divided into a few aliquots.

[25] The effective amount of administration of the active ingredient contained in the above composition can vary depending on the age, physical conditions, body weight, etc., of a patient, preferably 0.1 30 mg/kg/day, more preferably 0.5 3 mg/kg/day. The administration can be performed once or a few times per day within the range of the daily effective amount of administration.

[26]

[27] EXAMPLES

[28] The following examples illustrate the invention and are not intended to limit the same.

[29]

[30] Example 1: Confirmation of cytotoxicity of SMA

[31] The cytotoxicity of sodium meta-arsenite(NaAsO2, SMA) was examined by MTT assay using a cultured Huh-7 cell line. On the test day, Huh-7 cells were plated onto a 24- well plate to the final concentration of 8 10⁴ /well. On the next day, SMA was added at a concentration of 0, 0.1, 0.5, 1, 5, 10 M, respectively, and the MTT analysis was performed for 24, 48, 72 hours after the above addition.

[32] The MTT assay was performed three times per each sample and the mean value was obtained therefrom. The results are represented as a curve for Huh-7 cell growth inhibition according to various SMA concentration along the time passage(Fig. IA) and curve for cell viability(%) compared to that of control group according to time passage along various SMA concentration (Fig. IB).

[33] In addition, according to time passage, the cell status was taken pictures and cell nuclei were dyed using DAPI(4,6-diamidino-2-phenylindole) and then observed under fluorescent microscope. The result showed that in the cells treated with SMA at a concentration of 0.5 M or less there was no cytotoxicity observed but cell growth inhibition rate reached 40%. In the cells treated with SMA at a concentration of 5 M or higher cell growth was completely stopped due to the higher cytotoxicity (Fig. IA).

[34] The LD_50, the concentration of SMA when the cells treated with SMA for 3 days show 50% reduction in cell growth as compared to those not treated with SMA , was found to be about 3.5 M.

[35] According to the result of cells pictured 48 hours after SMA treatment, in the cells treated with 1 M of SMA , there was no noticeable cytotoxicity observed, whereas in the cells treated with 5 M or higher of SMA the presence of cytotoxicity was clearly observed (Fig. X).

[36] According to the cell nuclei staining with DAPI, there was only a negligible level of apoptosis in the cells treated with 1 M of SMA , but in the cells treated with 2.5 M of SMA there was observed a considerable level of apoptosis (Fig. 3).

[37]

[38] Example 2: Confirmation of the inhibitory activity of SMA in the hepatitis C virus

[39] The inhibitory activity of SMA against hepatitis C virus was measured in cultured cells.

[40] A full-length RNA of hepatitis C virus was introduced into Huh-7 cells by electric shock. When the virus proliferation observed under fluorescent microscope reached about 80%, the viral cells were treated with various concentrations of SMA . After the treatment, the degree of viral replication during 24, 48 and 72 hours were observed by means of immune-blotting of viral protein and NS5A (Figs. 4 & 5) and by im- munofluorscence staining (Fig. 6)

[41] Based on the above-mentioned cytotoxicity result, the effects of SMA in the range of

1 M or less, where the cytotoxicity is little or at a negligible level, were observed first (Fig. 4), and then, observed at a concentration where the cytotoxicity is of significance (Fig. 5). The change in the amount of NS5A detected by immuno-blotting is shown according to SMA concentration (Fig. 7A) and time treated (Fig. 7B).

[42] As a result, the cells treated with SMA at a concentration of 0.5 - 1 M for 24 hours showed 20 - 40% of antiviral activity, those at a concentration of 2.5 M for 24 hours showed 45 - 50% of antiviral activity, and those at a concentration of 5 - 10 M for 24 hours showed 50 - 70% of antiviral activity (Fig. 5).

[43] From the above, it was found that the half maximal inhibitory concentration of SMA is about 2.0 M (Fig. 7A). The cytotoxicity at this concentration is not high and thus the antiviral activities shown in the above test results appear to be largely due to the direct antiviral activities of SMA .

[44]

[45] Example 3: Subacute toxicity teston mice

[46] A subacute toxicity test for oral administration was performed by using five week- old specific pathogen free (SPF) CD- 1 mice.

[47] The SMA of the present invention was dissolved respectively in 10% glucose solution and orally administered once daily for 14 days at a concentration of 10, 20, 40 and 80 mg/kg/day per each group of mice, wherein each group consists of 10 mice. On the 29^th day from the above treatment, after allowing a recovering period of 14 days, biochemical test and hematologic test were performed for the mice and they were then autopsied and observed for the presence of any abnormalities in organs by naked eye. The mice, after the SMA treatment, were observed daily for the presence of any clinical symptoms, change in body weight, and their survival.

[48] As a result, it was found that all mice in the groups treated with 10, 20 and 40 mg/kg of SMA survived, no change in body weight and no noticeable clinical symptoms were observed. Further, no toxicity was observed in blood test, hematologic biochemical test and no toxicological changes were observed from autopsies.

[49] Meanwhile, of the mice in the group treated with 80 mg/kg of SMA three mice died on the 4^th day from the treatment, one died on the 7^th and four of the remaining six showed dyspnea but did not die.

[50] According to the subacute toxicity test of the present invention, CD- 1 mice orally administered with SMA did not show any toxicity up to 40 mg/kg of treatment thus showing that the safe dose for the oral administration of SMA is 40 mg/kg or less.

[51]

[52] Preparation Example 1: Preparation of Tablets

[53] 100 mg of sodium meta-arsenite (SMA) as an active ingredient was mixed with 40 mg of lactose, 51 mg of corn starch and 2 mg of colloidal silicon dioxide to prepare a tablet of 200 mg. In fact, the mixture was added with 3% polyvinyl pyrrolidone solution, pulverized and sieved with a size 14 sieve. The resultant was dried, sieved with a size 14 sieve and then added with 1 mg of magnesium stearate to obtain a tablet. [54]

[55] Preparation Example 2: Preparation of Injections

[56] 10 mg of sodium meta-arsenite (SMA) as an active ingredient was dissolved in 200 mg of polyoxyethylene hydrogenated caster oil and then added with water for injection to a final volume of 10 mL of an ampoule to prepare an injectable drug with a final concentration of 1 mg/mL.

Industrial Applicability

[57] The composition of the present invention comprises sodium meta-arsenite as an active ingredient which has excellent anti-HCV activity but with low cytotoxicity and is thus expected to be effective for the treatment of hepatitis C.

Claims

[1] A composition comprising sodium meta-arsenite as an active ingredient for the treatment of hepatitis C.