More particularly, the invention relates to a method for treating disease or disorder associated with inflammation comprising administering the byakangelicin into a subject, a method for inhibiting the production of TNF-a comprising administering the byakangelicin into a subject, a pharmaceutical composition comprising the byakangelicin and use thereof.

Background of the Invention Inflammation is a main defense response occurring in body against tissue injury and microbial infection. Inflammation accompanies systemic symptoms such as fever, weakness, loss of appetite, chill and pain of joint, and local symptoms such as redness, swelling, pain and loss of function. Such clinical symptoms are due to the change in blood vessels occurring during inflammation response. First, local blood flow is increased due to the expansion of diameter of blood vessels and accordingly, fever and redness are caused. In addition, endothelial cells constituting the inner wall of blood vessel are activated and increase the expression of adhesion molecules that promote the binding of circulating leukocytes. Lastly, permeability of blood vessels is changed and thus, endothelial cells wrapping blood vessel walls are separated therefrom and form the exit of liquids and proteins in

bloods, thereby inducing local accumulation thereof in tissues. This constitutes a cause of tumefaction, swelling and pain.

Such inflammation response causes numerous kinds of diseases. As main inflammation diseases, there are rhinitis and sinusitis such as infectious rhinitis, allergic rhinitis, chronic rhinitis, acute sinusitis and chronic sinusitis; otitis media such as acute purulent otitis media and chronic purulent otitis media; pneumonia such as bacterial pneumonia, bronchopneumonia, lobar pneumonia, Legionella pneumonia and viral pneumonia; acute or chronic gastritis; enteritis such as infectious enterocolitis, Crohn's disease, idiopathic ulcerative colitis and pseudomembranous colitis; and arthritis such as pyogenic arthritis, tuberculous arthritis, degenerative arthritis and rheumatoid arthritis.

Septic shock, one of the inflammation diseases, which accompanies extreme systemic inflammation response at early stage results from excessive systemic response of host cells against bacterial infection. In particular, this disease is characterized by shock phenomenon that occurs in organs after poisoning due to endotoxin of Gram-negative bacteria. Endotoxin induces disease by eliciting excessive response of immune system of host against it. The prognosis of septic shock is considerably serious. According to National Institute of Health in the USA, the death rate of patients due to septic shock amounts to approximately 30% and it has been a main cause of death, regardless of age ranging from infants to adults.

As agents which have been developed to treat disease or disorder associated with inflammation until now, there are antimicrobial and anti-inflammatory agents.

However, their effects are not good and they cause side effect. Accordingly, development of a new agent is continuously needed.

Meanwhile, all the changes associated with the above inflammation responses are induced by several kinds of inflammation mediators that are released after host cells recognized pathogens. As inflammattion mediators, there are prostaglandin, leukotriene and platelet-activating factor (PAF) and the like which are lipid mediators, and they are produced by macrophage. The response by such mediators is followed by the response by chemokine and cytokine that are produced and released by macrophage.

TNF-a, a representative cytokine of the inflammation mediators, is produced in fibroblast and macrophage which are activated by inflammation- inducing factor such as BCG (Bacille Calnette-Guerin) and LPS (lipopolysaccharides) and induces cell death and production of cytokine such as IL- 1 and IL-6. Since relationship between TNF-a that was found as tumor necrosis factor and inflammation diseases including rheumatoid arthritis, auto-immune diseases such as rejection of organ transplantation, asthma, allergic inflammatory diseases such as atopy dermatitis, and acute immune diseases such as sepsis and acute hepatic disorder was revealed, researches about compounds capable of inhibiting the synthesis of TNF-a have been under active progress. It is known that the mouse whose gene of TNF-a receptor is injured shows resistance to lethal toxicity by LPS and the mouse administered with antibody against TNF-a has an effect on preventing arthritis or toxicity of LPS (Beutler B., Science, 229: 869,1985 ; Pfeffer K et al., Cell, 73; 457,1993).

In connection with researches for developing medical products capable of inhibiting the production of TNF-a, it was reported that cytokines such as IL-4, IL- 10 and TGF-D (tumor growth factor-) have inhibitory activity against TNF-a,

and clinical experiments using TNF-a antibody and TNF-a receptor are being conducted. In addition, studies about pentoxifylline, thaliodomide, pyrrolidine dithiocarbamate, bicyclic imidazole and the like have been under progress, but so far, a compound capable of inhibiting the production of TNF-a has not been developed as a therapeutic agent (A. Eigler et al., Immunology Today, 18: 487-492,1997).

Meanwhile, byakangelicin is a known compound. Korean Patent Registration No. 139769 (March 5,1998) discloses the preventive effects on diabetic or senile cataract as a pharmaceutical use of byakangelicin. However, the fact that byakangelicin has prevention and treatment effects on inflammation disease has never been disclosed.

In the course of conducting studies to develop therapeutic agent for disease associated with inflammation having no side effect and high anti-inflammatory effect, the inventors found that byakangelicin has anti-inflammatory activity by inhibiting the production of TNF-a, which is different from its already-known therapeutic use and thus it can be used as a therapeutic agent for disease associated with inflammation, whereby the present invention has been completed.

Object of the Invention Accordingly, an object of the present invention is to provide a method for preventing and treating disease or disorder associated with inflammation comprising administering an effective amount of byakangelicin into a subject.

Another object of the invention is to provide a method for inhibiting the production of TNF-a comprising administering an effective amount of

byakangelicin into a subject.

Further, another object of the invention is to provide a pharmaceutical composition for preventing or treating disease or disorder associated with inflammation comprising an effective amount of byakangelicin.

Further, another object of the invention is to provide a pharmaceutical composition for inhibiting the production of TNF-a comprising an effective amount of byakangelicin.

Further, another object of the invention is to provide a use of byakangelicin for the preparation of an agent for preventing or treating disease or disorder associated with inflammation.

Further, another object of the invention is to provide a use of byakangelicin for the preparation of an agent for inhibiting the production of TNF-a.

Summary of the Invention Accordingly, the present invention provides a method for preventing and treating disease or disorder associated with inflammation comprising administering an effective amount of byakangelicin into a subject.

Also, the invention provides a method for inhibiting the production of TNF- a comprising administering an effective amount of byakangelicin into a subject.

Also, the invention provides a pharmaceutical composition for preventing or treating disease or disorder associated with inflammation comprising an effective amount of byakangelicin.

Also, the invention provides a pharmaceutical composition for inhibiting the

production of TNF-a comprising an effective amount of byakangelicin.

Also, the invention provides a use of byakangelicin for the preparation of an agent for preventing or treating disease or disorder associated with inflammation.

Also, the invention provides a use of byakangelicin for the preparation of an agent for inhibiting the production of TNF-a.

Detailed Description of the Invention The present invention will be described in detail.

The compound according to the present invention is byakangelicin represented by the following formula I: The byakangelicin may be isolated and purified from nature, commercially purchased, or prepared by chemical synthesis known in the pertinent art.

Preferably, the byakangelicin according to the present invention may be isolated and purified from nature. Most preferably, byakangelicin can be isolated and purified from Angelica dahurica (Benth, et Hooker). Angelica dahurica can be commercially easily purchased from medicinal herb markets. Isolation and purification of the compound according to the present invention from Angelica

dahurica can be carried out by the method known in the pertinent art (Kiyoshi Hata et al. , Yakugaku Zasshi. 83,606-610, 1963; Kuk Hyun Shin et al., Arch. Pharm. Res.

17, 331-336, 1994). Preferably, desired pure compound can be obtained by extracting an active ingredient of Angelica dahurica using a solvent and then by isolating and purifying it using chromatography.

In an embodiment of the invention, after rootstock of Angelica dahurica was extracted using 70% methanol, it was extracted by distribution extraction using water, n-hexane, ethyl acetate and butanol and concentrated under a reduced pressure to obtain fractions, and such fractions were purified using silica gel column chromatography to yield byakangelicin. Various kinds of organic solvents known in the pertinent art may be used, but a mixed solvent of n-hexane and ethyl acetate and a mixed solvent of ethyl acetate and methanol are preferably used.

The byakangelicin according to the present invention has preventive and therapeutic effects on disease or disorder associated with inflammation. The inventors used a septic shock animal model to measure medicinal effects of the compound of formula I.

The septic shock animal model is a known method to measure medicinal effects of a certain compound by administering septic shock-inducing factors such as bacteria themselves, LPS of bacteria or inflammatory cytokine to thereby induce a septic shock artificially (Deitch, Shock, 9; 1-11, 1998).

In the embodiment of the present invention, as septic shock animal model, a low-dose LPS mouse model was used, in which mouse was administered with LPS (lipopolysaccharide). When LPS is administered into mouse along with D-

galactosamine, sensitivity of mouse to LPS is remarkably increased, thereby inflammation is induced and septic shock is thus developed. The inventors administered byakangelicin into low-dose LPS mouse model and determined survival rate of mice, level of cytokines (TNF-a, IL-6 and IL-10) in bloods and hepatotoxicity value. As a result, the survival rate of mice was remarkably increased, the level of TNF-a was remarkably decreased, and the hepatotoxicity value was remarkably decreased. Accordingly, the inventors confirmed that the byakangelicin according to the present invention has an excellent effect on prevention or treatment of septic shock, which is a disease associated with inflammation; and inhibition of the production of TNF-a (see Example 2).

Accordingly, the present invention provides a method for preventing or treating disease or disorder associated with inflammation comprising administering an effective amount of byakangelicin into a subject.

Also, the present invention provides a method for inhibiting the production of TNF-a comprising administering an effective amount of byakangelicin into a subject.

Preferably, byakangelicin may be administered into a subject after further comprising pharmaceutically acceptable carriers and being formulated as a dosage form according to the known methods. The"pharmaceutically acceptable"carrier as used herein refers to a substance that is physiologically acceptable and does not cause allergy responses such as gastrointestinal disorder, dizziness and the like, or responses similar thereto when administered into humans.

The pharmaceutically acceptable carriers may further include, for example,

carriers for oral and parenteral administrations. The carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Also, the carriers for parenteral administration may comprise water, suitable oils, saline, water-soluble glucose and glycols and the like. In addition, the pharmaceutically acceptable carriers may further comprise stabilizers and preservatives. As a suitable stabilizer, there are antioxidants such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite. As suitable preservatives, there are benzalkonium chloride, methyl-or propyl-paraben and chlorobutanol. For other pharmaceutically acceptable carriers, the following literature can be consulted; Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995.

The byakangelicin according to the present invention can be formulated into various parenteral or oral dosage forms, together with the pharmaceutically acceptable carriers as described above. For example, a typical dosage form for parenteral administration is that for injection and preferably, an isotonic aqueous solution or a suspension. The dosage form for injection can be prepared using a suitable dispersion agent, wetting agent and suspension agent according to the known methods in the pertinent art. For example, each ingredient is dissolved in saline or buffer and then can be prepared into a dosage form for injection. Also, as a dosage form for oral administration, for example, there are powders, granules, tablets, pills, capsules, etc. , which may comprise diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine) and/or lubricants (such as silica, talc, stearic acid and magnesium or calcium salt thereof and/or polyethylene

glycol) as well as the active ingredient. Furthermore, the tablets may further comprise binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine and if necessary, they may further comprise disintegrants or effervescent mixtures such as starch, agar, alginic acid or sodium salt thereof, and/or absorbents, colorants, flavors and sweeteners. Such dosage forms can be prepared by conventional mixing, granulation or coating methods.

The pharmaceutical composition formulated according to the above method can be administered into a patient via several routes including oral, percutaneous, subcutaneous, venous or muscular routes.

The"effective amount"as used herein refers to an amount of the compound capable of having preventive or therapeutic effects on disease or disorder associated with inflammation or inhibiting the production of TNF-a when administered into a subject. Preferably, its ordinary dose per day is within the range of 10 to 1,000 mg/kg. The byakangelicin may be administered once or more times a day within the preferred dose. The dose of the byakangelicin according to the present invention may be suitably selected according to administration route, subject, age, sex, body weight, individual variation and condition of disease.

The"subject"as used herein refers to mammals including humans and preferably humans diagnosed as disease or disorder associated with inflammation.

The"disease or disorder associated with inflammation"as used herein includes all diseases or disorders induced by inflammation response occurring in body. For example, they include, but are not limited to, inflammatory intestine diseases such

as Crohn's disease and ulcerative colitis, peritonitis, osteomyelitis, cellulitis, meningitis, cerebritis, pancreatitis, trauma-inducing shock, bronchial asthma, allergic rhinitis, cystic fibrosis, cerebral apoplexy, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, hepatitis, nephritis, osteoarthritis, gout, spinal arthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with'vasculitis syndrome', polyarteritis nodosa, hypersensitive vasculitis, Wegener's granulomatosis, polymyalgia rheumatica, giant cell arteritis, calcium crystal deposition arthropathy, pseudo gout, non-joint rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schonlein purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytoma, scoliosis, hemochromoatosis, meniscocytosis, other hemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinaemia, hyperparathyroidism, acromegaly, familial mediterranean fever, Gerhardt disease, systemic lupus erythematosus, relapsing fever, psoriasis, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ dysfunction syndrome, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, broncho-pulmonary dysplasia and the like.

The byakangelicin of the present invention for preventing or treating disease or disorder associated with inflammation and inhibiting the production of TNF-a can

be used alone in treatment of the disease or disorder as described above, or can be used in admixture with other pharmaceutical effective ingredients including lipid- lowering agents, cholesterol-lowering agents, HMG-Co A reductase (3-hydroxy-3- methylglutaryl coenzyme A reductase) inhibitors, myocardial protectants, therapeutic agents for coronary artery disease, other therapeutic agents for hypertension, therapeutic agents for chronic heart failure, therapeutic agents for diabetes, other improving agents for insulin sensitivity, therapeutic agents for hypothyroidism, therapeutic agents for nephrotic syndrome, anti-inflammatory agents, therapeutic agents for bone diseases (therapeutic agents for osteoporosis and the like) or therapeutic agents for chronic renal failure.

The invention provides a pharmaceutical composition for preventing or treating disease or disorder associated with inflammation comprising administering an effective amount of byakangelicin.

The invention provides a pharmaceutical composition for inhibiting the production of TNF-a comprising administering an effective amount of byakangelicin.

These pharmaceutical compositions may further comprise pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carriers are as described above. The pharmaceutical composition according to the present invention can be formulated as a dosage form according to the methods known in the pertinent art so that after being administered into a subject, it can release the active ingredient in a prompt, continuous or prolonged way.

In addition, the invention provides a use of byakangelicin for the preparation of an agent for preventing or treating disease or disorder associated with inflammation.

Also, the invention provides a use of byakangelicin for the preparation of an agent for inhibiting the production of TNF-a.

The agent comprising the byakangelicin may further comprise pharmaceutically acceptable carriers and be formulated as a suitable form according to the known methods. Examples of disease or disorder associated with inflammation are as described above.

The present invention will be described on the basis of the following examples in more detail. However, the Examples shown below are provided solely to illustrate the invention and do not limit the invention in any way.

EXAMPLE 1 Isolation and Purification of Byakangelicin from Rootstock of Angelica dahurica In order to investigate novel anti-inflammatory activity of byakangelicin, byakangelicin was isolated and purified from rootstock of Angelica dahurica.

That is, 9.6 kg of root of dried Angelica dahurica was extracted with 10Q of mixed solution of methanol and water (7: 3) three times for 12 hours each time while being refluxed, cooled at 60 °C. The obtained extract was filtrated under a reduced pressure using a vacuum concentrator and concentrated to thereby obtain 3 kg of

methanol extract. A mixed solution of 1. 5Q of water and 1. 59 of n-hexane was added to the above methanol extract while distribution extraction into n-hexane layer and water layer was carried out using fraction funnel two times. The n- hexane layer was concentrated under a reduced pressure to obtain 710 g of n-hexane extract, and the water layer was extracted with ethyl acetate and butanol in turn according to the above method to thereby obtain 35.8 g of ethyl acetate extract, 128.95 g of n-butanol extract and 2.1 kg of water extract.

After that, silica gel column chromatography of 35.8 g of ethyl acetate extract was carried out. That is, column (80 cm in length, 8 cm in diameter) was filled with silica gel (No. 9385,230-400mesh, Merck) up to about 21 cm, it was eluted using 1Q of n-hexane: ethyl acetate (6: 4) mixed solvent as a developing solvent, thereby saturating stationary phase and then, 35.8 g of ethyl acetate extract was adsorbed on about 45 g of silica gel (No. 7734,70-230 mesh, Merck), thereby being loaded onto the column. Mobile phase elution was started using a mixed solvent of n-hexane: ethyl acetate (6: 4), and fractions 1 to 16 were separated by flash column chromatography method wherein the polarity of ethyl acetate: methanol mixture was gradually raised and at the same time, N2 gas was flowed to the column at a certain rate. Among such fractions, fraction 11 was dissolved in a mixed solution of n-hexane: ethyl acetate (6: 4) and then placed at room temperature to thereby yield a crystalline substance.

Physicochemical and spectroscopic properties of such isolated and purified substance were determined and the results are as shown below. According to the results, the substance purified from fraction 11 was identified as byakangelicin (Kiyoshi Hata et al., Yakugaku Zasshi. 83,606-610, 1963; Kuk Hyun Shin et al., Arch. Pharm. Res. 17,331-336, 1994). p 118^C IR v max (KBr, cm1) : 3540, 3100, 3120, 1710, 1605, 1595, 1490, 1386, 1370, 820 US (n/z) : 334 (4), 319(M.-Me). 245(M.-C.H@O2). 232 (W+C5H10O2), 217 (232-Me). 203 (231-CO), 189 (217-CO). 175 (203-00).

160 (189-CHO) , H-NMR (CDCl5) # : 6. 27 (IH. d, J=9. 8, H-4). 7.00 91H, d. J=2. 4. H-2) 7. 63(1H,d, J=2. 4. H-3'). 4.14- 4. 36 (2H. m. H-a). 4. 60 (1H, dd. H-b). 1.31 (3H. s. H-d), 1. 27(3H,s,H-O@). 4. 17(3H,S,OMe).

EXAMPLE 2 Test of Effects of Byakangelicin In the present invention, a low-dose LPS model was used to measure anti- inflammatory activity of byakangelicin, in which septic shock was caused by intraperitoneally administering ICR mice (20-30 g in body weight, 4-6 weeks old) with 36 ag/kg of LPS and 0.8 g/kg of D-galactosamine.

The 20 ICR mice were divided into four groups of five mice. At 30 min. before the administration of LPS and D-galactosamine, mice in the above three groups were intraperitoneally administered with byakangelicin, which was prepared in above Example 1, dissolved in 10% solution of DMSO (dimethylsulfoxide) in dosages of 10mg/kg, 30mg/kg and 100mg/kg, respectively (experimental group 1, experimental group 2 and experimental group 3, respectively), and mice in the remaining one group (control group) were intraperitoneally administered only with 10% DMSO solution of the same amount (0.1 ml).

2-1) Measurement of Survival Rate of Mice

Survival rates of mice in the above experimental group 1, experimental group 2, experimental group 3 and control group were determined over the lapse of time, and the results are shown in Table 1.

Table 1 Survival Rate Survival Rate Survival Rate After 24 hours (%) After 48 hours (%) After 72 hours (%) Control Group 20 20 20 Experimental 80 80 80 Group 1 Experimental 100 100 100 Group 2 Experimental 80 80 80 Group 3

As shown in Table 1 above, in all of experimental groups 1,2 and 3 where byakangelicin was administered, death rate due to septic shock was remarkably decreased as compared with the control group and especially, in the case that the byakangelicin was administered in an amount of 30 mg/kg (experimental group 2), all mice survived. According to the above result, it could be seen that the byakangelicin is very effective in prevention or treatment of diseases or disorders associated with inflammation, especially, septic shock.

2-2) Level Measurement of TNF-a, IL-6 and IL-10 as Cytokines Bloods were collected from mice in the above experimental group 2 and control group at 90 min. , 3 hours and 6 hours, respectively after intraperitoneal administration of LPC/D-galactosamine. After serum was isolated from the above- collected bloods using centrifuge, the levels of TNF-a, IL-6 and IL-10 in the above

serum were each determined using ELISA kit. The results are shown in Fig. 1 a to Fig. Ic.

As shown in Fig. la, in bloods in the mice of experimental group 2, TNF-a which is an inflammation-inducing cytokine, was remarkably decreased as compared with the control group. On the other hand, the levels of IL-6 and IL-10 in the bloods of experimental group 2 showed no significant change (see Fig. lb and Fig. lc). According to the above results, it could be seen that byakangelicin can effectively prevent and treat inflammatory diseases by decreasing the level of TNF- a, which is a key factor associated with inflammatory diseases, in bloods.

2-3) Hepatotoxicity Test 0.5 m of blood was collected from auricle vein of mice in the above experimental group 2 and control group, respectively at 90 min. after intraperitoneal administration of LPC/D-galactosamine. After serum was isolated from the collected blood using centrifuge, the level of ALT (alanine aminotransferase ; also called GPT (glutamic-pyrubic transaminase)), which is an index of hepatotoxicity, was measured according to Reitman-Frankel method (Reitman S. and Frankel S., Am. J. Clin. Pathol. 28: 56-63,1957), and the results are shown in Fig. 2.

As shown in Fig. 2, the ALT value, which is the hepatotoxic index, in the experimental 2 was remarkably decreased as compared with the control group.

According to the above results, it could be seen that byakangelicin has an effect on prevention and treatment of septic shock due to inflammation.

As shown in the results of the above Examples, it was proven that the

composition comprising byakangelicin according to the present invention can remarkably decrease death rate due to septic shock, concentration of TNF-a in blood and hepatotoxicity and accordingly, byakangelicin can be used as a novel use, that is, preventive and therapeutic agent for diseases associated with inflammation.

Industrial Applicability Byakangelicin according to the present invention can be effectively used in prevention or treatment of disease or disorder associated with inflammation, especially septic shock, and inhibition of the production of TNF-a