KR100285586B1 - Agent for treatment of b-type hepatitis containing 5-hydroxymethyl-2-furfural - Google Patents

Abstract

PURPOSE: A medicament for treating hepatitis B containing 5-hydroxymethyl-2-furfural exhibiting an inhibiting effect on the formation of a hepatitis B virus surface antigen (HBVsAg) and the DNA formation of Hepatitis B virus (HBV) as an active component is provided. The 5-hydroxymethyl-2-furfural shows low toxicity as compared to Ara-AMP. CONSTITUTION: 5-Hydroxymethyl-2-furfural originated from natural drug, food and chemical synthetics has a therapeutic effect on hepatitis B. The compound can be mixed with conventional excipients such as filler, coagulation preventing agent, lubricant, flavoring agent and the like by conventional methods and formulated in the form of an agent for oral administration, hypodermic injection and intravenous injection.

Description

Hepatitis therapies containing 5-hydroxymethyl-2-furfural

Figure 1 shows the results of comparative analysis of the inhibition of hepatitis B virus DNA production by the comparative quantitative PCR method of 5-hydroxymethyl-2-furfural and the standard Ara-AMP.

The present invention relates to the use of 5-hydroxymethyl-2-furfural as a therapeutic agent for hepatitis B. More specifically, it was confirmed that 5-hydroxymethyl-2-furfural exhibits a strong inhibitory effect on hepatitis B surface antigen and hepatitis B virus DNA production, and using 5-hydroxymethyl-2-furfural It offers the possibility to develop treatments for hepatitis B.

Hepatitis B virus (“HBV”) is a virus of the Hepadnaviridae strain that specifically infects the human body. Around 250 million people worldwide are infected with HBV. The hepatitis B virus is distributed in various parts of the world, but the incidence rate varies greatly from country to country, with the US at 0.5%, Japan at 1.5%, Europe at 1%, 8% in Korea, 15% in China and Africa. The incidence rate is as high as 7%. To date, the Korean population has about 8% hepatitis B virus, of which 15 to 20% are known to develop cirrhosis, and about 20% of cirrhosis patients develop liver cancer. It is estimated that patients with cirrhosis and 140,000 liver cancers will occur (IH Hu et al., Monthly Pharmacy April, pp. 54-59 (1992)).

The incubation period of the hepatitis B virus is about 60 to 110 days, and 90 to 95% of the hepatitis B virus is completely recovered through various clinical stages.

In patients who have not recovered from infection, HBV DNA is assimilated into genomic DNA of human hepatocytes, leading to chronic active hepatitis, cirrhosis, liver cancer, and the like. Chronic hepatitis caused by HBV, like other diseases, causes chronic viral infections, lymphoma diseases and chronic kidney disorders. Therefore, chronic hepatitis is a disease with a high mortality rate that develops into a more powerful form of disease and eventually leads to death (Don Ganem et al, Ann. Rev. Biochem. 56, 651 (1987); RP Beasley et. al., Lancet 2, 1129 (1981); DA Shafritz et al., New England J. of Medicine 305, 1067 (1981); SN Zaman et al., Lancet 1, 1357 (1985)).

To date, many vaccines have been developed to prevent HBV infection. These vaccines use type B surface antigen (HBS Ag) as an immunogen. Hepatitis B surface antigen can be obtained from the plasma of carriers infected with hepatitis B virus or through genetic engineering techniques. Such surface antigenic vaccines are generally effective in preventing infection of individuals, but not all levels of protective antibodies are formed in all individuals to which the vaccine is administered. The degree of formation of these antibodies is influenced by factors such as the injection site as well as the age of the subject to which the vaccine is administered and the degree of suppression of the immune system. In addition, everyone who is given the vaccine has some risk of developing a hypersensitivity reaction.

Once infected, adequate treatment is required. Although several drugs have been developed as treatments for hepatitis B, few have satisfactory efficacy. Ara-A (Vidarabine) is by far the only drug approved by the US FDA.

Ara-A has shown significant therapeutic effects in clinical trials, but its use is extremely limited due to the toxicity of the drug itself and is used in combination with interferon. Side effects include nausea, loss of appetite, diarrhea, vomiting and reversible bone marrow suppression due to thrombocytopenia (J.A. Payne et al., Disease a Month, March, pp, 117-159 (1988)). Acyclovir (9- (2-hydroxyethoxymethyl guanine)) or interferon, on the other hand, shows some therapeutic effects on hepatitis B during administration, but once it is stopped, it returns to its pre-administration status. Long-term dosing because it returns, there is a problem that causes severe side effects.

Other promising phosphonoformates (Phosphonoformate, Foscarnet), Suramin, Thymosin, Prostaglandin, 2-carbocyclic analogue of 2-deoxyguanosine, Famciclovir, etc. Although it is known that this method requires more verification. FIAU (Fialuridine), one of the most promising drugs in terms of efficacy and toxicity, has been discontinued due to unexpected death in Phase II clinical trials.

Until now, studies on hepatitis B treatment have been predominantly related to nucleoside analogues, but severe side effects of these analogues are pointed out as the biggest disadvantages. On the other hand, studies have been reported to develop a hepatitis B therapeutic agent from natural drugs that are not nucleoside analogs. Extracts of medicinal plants, Phyllanthus niruri and Phyllanth-thus amarus, widely known as treatments for Indian jaundice, have been reported to be very effective in the inhibition of hepatitis B virus in animal experiments. In analysis and clinical trials (PS Venkateswaran et al., Proc. Natl. Acad. Sci. 84, 274 (1987); BS Blumberg et al., Vaccine 8, 86 (1990)).

Accordingly, the present inventors have also confirmed that the water-soluble extract of pollen has an inhibitory effect on the production of hepatitis B virus while trying to find a hepatitis B therapeutic agent from natural medicine (see the same patent application).

Furthermore, the present inventors have isolated and purified a single effective ingredient from this water-soluble pollen extract, and as a result of NMR structure analysis, it was confirmed that pyridoxine-HC1 and 5-hydroxymethyl-2-furfural are active ingredients (see the same patent application No. ).

5-hydroxymethyl-2-furfural is present in fruit juices, foods, liquor, etc. and is known to be easily made from polysaccharides such as sugar (Chemical Abstract 77, 85191a; 82, 18916v; 85, 44835k; 81, 39339q; 82, 113463q; 76, 12684p), dialdehydes, glycol aminoalcohols, acetals and the like.

However, there have been no studies on the biological activity of this substance, and no research has been made on its potential as a treatment for hepatitis B.

Therefore, the present inventors conducted a more careful study on the possibility of 5-hydroxymethyl-2-furfural as a therapeutic agent for hepatitis B based on the results of the above pollen extract, and the substance is hepatitis B virus and hepatitis B. The present invention has been shown to exhibit a strong inhibitory effect on the surface antigen production.

It is therefore an object of the present invention to provide a hepatitis B therapeutic agent comprising 5-hydroxymethyl-2-furfural.

Hereinafter, the present invention will be described in detail.

After treatment with an appropriate amount of 5-hydroxymethyl-2-furfural in a cell line producing hepatitis B virus and incubating for a predetermined time, cell culture medium and cells are collected to generate hepatitis B surface antigen and to generate hepatitis B virus DNA. By analyzing the degree of inhibition it can be seen that 5-hydroxymethyl-2furfural can be used as a treatment for hepatitis B.

5-Hydroxymethyl-2-furfural may be used by any method derived from natural medicines, food-derived, chemical compounds, etc. In the embodiment of the present invention, 5-hydroxymethyl- purchased from Aldrich. 2-furfural (# H4080-7) is used.

Inhibitory concentrations for surface antigens are analyzed using a commercialized hepatitis B surface antigen detection kit (Auszyme®, Monoclonal, Abbott 1ab., # 1980-24).

The degree of inhibition against hepatitis B virus DNA was determined by comparative quantitative PCR method (R.K. Saiki et al., Science 239,487 (1988); M. Escarceller et al., Anal. Biochem. 206, 36 (1992)); S. Diviacco et al., Gene 122, 313 (1992). In this case, the HBV primers used are 3′-terminal primers between the nuclear antigen gene and the polymerase. It is used as a '-terminal primer and HBV as an index of PCR reaction using Trp R (RP Gunsalus and C. Yanofsky, PNAS 77, No 22,7117 (1980)), which is an E. coli gene as an internal index of PCR reaction. The genes of 320 bp and Trp R 450 bp are amplified and shown. DNA amplified by the quantitative PCR method is confirmed by electrophoresis on 7% polyacrylamide gel and then stained with silver.

As a result, 5-hydroxy-2-furfural has a strong inhibitory effect on the DNA production of hepatitis B surface antigen and hepatitis B virus, so 5-hydroxymethyl-2-furfural can be used as a therapeutic agent for hepatitis. It can be confirmed.

The hepatitis B therapeutic agent containing 5-hydroxymethyl-2-furfural as an active ingredient may include a pharmaceutically acceptable carrier and the like. The hepatitis B treatment agent can be administered intravenously or intramuscularly, and the daily dose is 5-15 mg / kg body weight, preferably 5 mg / kg body weight, but can be adjusted according to various conditions such as the patient's symptoms, age and sex. It is possible.

The hepatitis B therapeutic agent of the present invention may be formulated in the form of oral preparations, subcutaneous injections, intravenous injections, etc., in combination with conventional excipients, for example, fillers, anticoagulants, lubricants, flavoring agents, etc. Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are given for illustrative purposes only and in no way are the scope of the present invention limited thereto.

EXAMPLE

1. Cell Culture and Treatment of 5-hydroxymethyl-2-furfural

2.2.15 cells (MA Shells et al., PNAS 84,1005 (1987)), a cell line producing hepatitis B virus, were treated with 10% FBS (Fetus Bovine Serum: GIBCO, # 16000-028), 1% in a T75 flask. In IMDM medium (GIBCO Cat No. 430-2200) added ABAM (Antibiotic-Antimycotic, GIBCO BRL, # 15240-013), Geneticin (Sigma, # G-9516) at a final concentration of 400 μg / ml After incubation for 5 days, the cells were aliquoted to 1.0-1.5 × 10 ⁵ cells / ml / pore in a 24-hole microplate and incubated for 1-2 hours in a 5% CO ₂ incubator. After cells have adhered to the bottom, 100 μM of 5-hydroxymethyl-2-furfural is serially diluted with 10 ⁿ or 5 ⁿ (where n is 0, 1, 2, 3, 4) with the medium, 0.1 for each pore. Each ml was treated. At this time, the standard Ara-AMP (Sigma, # A-5392) was used in the same dilution. We tried to reduce the experimental error to the maximum by maintaining the number of repetitions for each sample. Cultures and culture cells were collected 6-8 days after sample treatment.

2. Analysis of Inhibition of Hepatitis B Surface Antigen

Hepatitis B surface antigen production of 5-hydroxymethyl-2-furfural using the cultured hepatitis B surface antigen detection kit (Auszyme R, Monoclonal, Abbott 1ab., # 1980-24). The degree of inhibition against was quantitatively analyzed.

Cell cultures were diluted with phosphate buffered saline (pH 7.0) to a final dilution in the range of 1.0 to 1.5 (approximately 20 to 50 fold) with 0.2 ml of diluent and Ara-AMP and 5-hydroxymethyl-2-furfural. 0.2 ml of the untreated control was reacted with the beads bound to the primary monoantibody against the surface antigen and the secondary monoantibody linked with horse radish peroxidase for 2 hours at 40 ° C., and then the beads were removed to remove unbound material. Was washed three times with distilled water. Thereafter, 300 µl of o-phenylenediamine solution containing hydrogen peroxide solution was added to the kit, followed by incubation for 30 minutes, and then 0.1N sulfuric acid solution was added to stop the reaction.

Absorbance of control and sample samples was measured with a spectrophotometer equipped at 492 nm, and the degree of inhibition of hepatitis B surface antigen production of 5-hydroxy-2-furfural and standard Ara-AMP from the measured absorbance was determined according to the following equation. The following results were obtained.

(At this time, the absorbance of the control is 1.023)

As can be seen from the above, the degree of inhibition of hepatitis B surface antigen of 5-hydroxymethyl-2-furfural was almost equivalent to that of the standard Ara-AMP.

3. Analysis of the degree of inhibition of hepatitis B virus DNA production by comparative quantitative PCR

Comparative Quantitative PCR Methods (RK Saiki et al., Science 239,487 (1988); M. Escarceller et al., Anal. Biochem. 206, 36 (1992); S. Diviacco et al., Gene 122, 313 (1992)) The degree of inhibition of hepatitis B virus DNA production was analyzed.

Each of the cultured cells of step 1 was treated with 0.5 ml of crushed solution (0.1N NaOH, 0.1% SDS), incubated at 65 ° C. for 30 minutes, extracted with a conventional method through phenol / chloroform treatment, and then tripled in volume. 100% ethanol was added and centrifuged at 14000 rpm at 4 ° C. Precipitated DNA (hereinafter referred to as “purified DNA”) was dried and dissolved in 100 μl of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA).

Meanwhile, the following primers were synthesized by a gene synthesizer (Applied Biosystem, model 380A, USA) using an automated solid phase phosphoamidite chemistry to amplify the purified DNA by PCR. It was.

Primers IntCP 5′- GAT AGG ATA GGG GCA TTT GGT GGT-3 ′

The primer is a 3′-terminal primer as a nucleotide sequence adjacent to a nuclear gene and a polymerase gene of hepatitis B virus.

Primer HBV200 5′-CGC CTC AGC TCT GTA TCG A-3 ′

The primer is a 5′-terminal primer as the nucleotide sequence upstream about 320bp from the primer IntCP.

Primers TrpR5 5′-GTA ATC TAG AGG GTA CAT ATT ATG GCC CAA CAA T-3 ′

The primer is a nucleotide sequence ranging from 21 bp upstream to 10 bp downstream of ATG, the translation start codon of the TrpR gene.

Primers TrpR3 5′-TAT TAA GCT TAC GGG TAT TTG TAG GAC GGA T-3 ′

The primer is a nucleotide sequence ranging from about 100 bp to 70 bp downstream of TGA, the translational stop codon of the TrpR gene.

5 μl of purified DNA and 1 μl (100 pg) of Trp R gene DNA in 5 μl of 10 × Taq DNA polymerase buffer [100 mM Tris-Hcl (pH 8.3), 500 mM KCl, 15 mM MgCl ₂ , 0.1% (W / V ) Gelatin] and 5 μl of dNTP ′s mixed solution (dGTP, dATP, dTTP, dCTP 2.0mM each), 2 μl TrpR5, 2 μl TrpR3, 2 μl HBV200, 2 μl IntCP, 25.8 μl distilled water, 0.2 μl of Taq DNA polymerase (5 u / λ, Boehringer Mannheim, Germany) was added and mixed well.

In order to prevent evaporation of the solution, 50 µl of mineral oil was added to the reaction solution and the temperature cycle program was performed at 94 ° C for 1 minute, 50 ° C for 1 minute, and 72 ° C for 1 minute using a thermocycler (Hybaid, Thermal Reactor, England). The nucleic acid amplification reaction was performed by repeating 5 times, 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 1 minute. The two amplified genes were electrophoresed on 7% polyacrylamide gel (TBE buffer, 7% acrylamide) and then stained silver. The results are shown in FIG.

In FIG. 1, the first to the fifth lanes show cultured cells treated with Ara-AMP, which is a standard product, by serial dilution (n = 0,1,2,3,4) at a concentration of 100 μM by 5 ⁿ . Lanes 7 to 11 show cultured cells treated with 5-hydroxymethyl-2-furfural by serial dilution (n = 0, 1, 2, 3, 4 ⁾ at a concentration of 100 μM by 5 ⁿ . Lanes 6 and 12 show cultured cells that were not treated with 5-hydroxymethyl-2-furfural or Ara-AMP at all.

The degree of inhibition of the production of hepatitis B virus DNA of 5-hydroxymethyl-2-furfural was almost the same as that of the standard Ara-AMP, showing a very strong inhibitory effect.

4. Toxicity test

After culturing 2.2.15 cells in IMDM medium as in 1, divide into 1.0 × 10 ⁵ cells / ml / hole and incubate for 1 to 2 hours. When the cells are completely attached to the bottom, three concentrations of 10mM, 1mM and 0.1mM After treatment with 5-hydroxymethylfurfural and Ara-AMP, after 3 days, the number of cells attached to each ball was counted according to the following formula, and the results were obtained as follows.

As can be seen from the above results, Ara-AMP showed 90% at 1 mM and 10% at 0.1 mM, but 5-hydroxymethyl-2-furfural showed about 5% at 1 mM.

This suggests that 5-hydroxy-2-furfural is 18 times less toxic than the standard Ara-AMP.

Claims (2)

A hepatitis B therapeutic agent comprising 5-hydroxymethyl-2-furfural as an active ingredient. The hepatitis B therapeutic agent according to claim 1, which is derived from 5-hydroxymethyl-2-furfural natural medicine, food-derived or chemically synthesized.